Icos ligand variant immunomodulatory proteins and uses thereof

ABSTRACT

Provided herein are immunomodulatory proteins comprising ICOSL variants and nucleic acids encoding such proteins. The immunomodulatory proteins provide therapeutic utility for a variety of immunological and oncological conditions. Compositions and methods for making and using such proteins are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional application No.62/323,608 filed Apr. 15, 2016, entitled “ICOS Ligand VariantImmunomodulatory Proteins and Uses Thereof,” U.S. provisionalapplication No. 62/394,745 filed Sep. 14, 2016, entitled “ICOS LigandVariant Immunomodulatory Proteins and Uses Thereof, ” U.S. provisionalapplication No. 62/410,842 filed Oct. 20, 2016, entitled “ICOS LigandVariant Immunomodulatory Proteins and Uses Thereof, ” U.S. provisionalapplication No. 62/472,568 filed Mar. 16, 2017, entitled “ICOS LigandVariant Immunomodulatory Proteins and Uses Thereof, ” and U.S.provisional application No. 62/475,162 filed Mar. 22, 2017, entitled“ICOS Ligand Variant Immunomodulatory Proteins and Uses Thereof,” thecontents of each of which are incorporated by reference in theirentirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled761612000300SeqList.txt, created Apr. 13, 2017, which is 979,474 bytesin size. The information in the electronic format of the SequenceListing is incorporated by reference in its entirety.

FIELD

The present disclosure relates to therapeutic compositions formodulating immune response in the treatment of cancer and immunologicaldiseases. In some aspects, the present disclosure relates to particularvariants of ICOS Ligand (ICOSL) that exhibit improved binding affinityfor one or both of the cognate binding partner proteins ICOS or CD28.

BACKGROUND

Modulation of the immune response by intervening in the processes thatoccur in the immunological synapse (IS) formed by and betweenantigen-presenting cells (APCs) or target cells and lymphocytes is ofincreasing medical interest. Mechanistically, cell surface proteins inthe IS can involve the coordinated and often simultaneous interaction ofmultiple protein targets with a single protein to which they bind. ISinteractions occur in close association with the junction of two cells,and a single protein in this structure can interact with both a proteinon the same cell (cis) as well as a protein on the associated cell(trans), likely at the same time. Although therapeutics are known thatcan modulate the IS, improved therapeutics are needed. Provided areimmunomodulatory proteins, including soluble proteins or transmembraneimmunomodulatory proteins capable of being expressed on cells, that meetsuch needs.

SUMMARY

In some embodiments, provided herein is a variant ICOS Ligand (ICOSL)polypeptide, comprising an IgV domain or specific binding fragmentthereof, an IgC domain or specific binding fragment thereof, or both,wherein the variant ICOSL polypeptide contains one or more amino acidsubstitutions in an unmodified ICOSL or a specific binding fragmentthereof corresponding to position(s) selected from 10, 11, 13, 16, 18,20, 25, 27, 30, 33, 37, 38, 42, 43, 47, 52, 54, 57, 61, 62, 67, 71, 72,74, 75, 77, 78, 80, 84, 89, 90, 92, 93, 94, 96, 97, 98, 99, 100, 102,103, 107, 109, 110, 111, 113, 115, 116, 117, 119, 120, 121, 122, 126,129, 130, 132, 133, 135, 138, 139, 140, 142, 143, 144, 146, 148, 151,152, 153, 154, 155, 156, 158, 161, 164, 166, 168, 172, 173, 175, 190,192, 193, 194, 198, 201, 203, 207, 208, 210, 212, 217, 218, 220, 221,224, 225, or 227 with reference to SEQ ID NO:32. In some embodiments,the unmodified ICOSL is a mammalian ICOSL or a specific binding fragmentthereof. In some embodiments, the unmodified ICOSL is a human ICOSL or aspecific binding fragment thereof. In some embodiments, the unmodifiedICOSL includes (i) the sequence of amino acids set forth in SEQ IDNO:32, (ii) a sequence of amino acids that has at least 95% sequenceidentity to SEQ ID NO:32; or (iii) a portion thereof comprising an IgVdomain or IgC domain or specific binding fragments thereof or both.

In some embodiments of any one of the variant ICOSL polypeptidesdescribed above, the specific binding fragment of the IgV domain or IgCdomain has a length of at least 50, 60, 70, 80, 90, 100, 110 or moreamino acids; or the specific binding fragment of the IgV domain includesa length that is at least 80% of the length of the IgV domain set for asamino acids 19-129 of SEQ ID NO:5 and/or the specific binding fragmentof the IgC domain includes a length that is at least 80% of the lengthof the IgC domain set forth as amino acids 141-227 of SEQ ID NO:5. Insome embodiments, the variant ICOSL polypeptide includes up to 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 aminoacid modifications, optionally amino acid substitutions, insertionsand/or deletions. In some embodiments, the ICOSL variant includes asequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity toSEQ ID NO:32 or a specific binding fragment thereof. In someembodiments, any of the variant ICOSL polypeptides provided hereinexhibit altered binding to the ectodomain of ICOS, CD28, or CTLA-4compared to the unmodified ICOSL. In some embodiments, any of thevariant ICOSL polypeptides exhibit altered binding to the ectodomain ofICOS or CD28 compared to the unmodified ICOSL. In some embodiments, thealtered binding is altered binding affity and/or altered bindingselectivity.

In some embodiments of any one of the variant ICOSL polypeptidesdescribed above, the one or more amino acid substitutions are selectedfrom M10V, M10I, V11E, S13G, E16V, S18R, A20V, 525G, F27S, F27C, N30D,Y33del, Q37R, K42E, T43A, Y47H, N52H, N52D, N52S, N52Q, N52Y, N52K,S54A, S54P, N57Y, N57D, R61S, R61C, Y62F, L67P, A71T, G72R, L74Q, R75Q,D77G, F78L, L80P, N84Q, D89G, E90A, K92R, F93L, H94E, H94D, L96F, L961,V97A, L98F, S99G, Q100R, Q100K, Q100P, L102R, G103E, V107A, V1071,S109G, 5109N, V110D, V110N, V110A, E111del, T113E, H115R, H115Q, V116A,A117T, N119Q, F1201, F1205, S121G, V122A, V122M, S126T, S126R, H129P,5130G,5132F, Q133H, E135K, F138L, T1395, C140D, C140del, S142F, I143V,I143T, N144D, Y146C, V151A, Y152C, Y152H,W153R, I154F, N155H, N155Q,K156M, D158G, L161P, L161M, L166Q, N168Q, F1725, L1735, M175T, T190A,T1905, S192G, V193M, N194D, C198R, N201S, L203P, L203F, N207Q, L208P,V210A, S212G, D217V, I218T, 1218N, E220G, R221G, R221I, I224V, T225A,N227K or a conservative amino acid substitution thereof.

In some embodiments, the one or more amino acid modifications areselected from among N52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P,N525/Y146C/Y152C, N52H/C198R, N52H/C140D/T225A, N52H/C198R/T225A,N52H/K92R, N52H/599G, N57Y/Q100P, N52S/G103E, N52S/S130G/Y152C,N52S/Y152C, N52S/C198R, N52Y/N57Y/Y152C, N52Y/N57Y/ H129P/C198R,N52H/L161P/C198R, N52S/T113E, N52D/S54P, N52K/L208P, N52S/Y152H,N52D/V151A, N52H/I143T, N52S/L80P, F120S/Y152H/N201S, N52S/R75Q/L203P,N52S/D158G, N52D/Q133H, N52S/N57Y/H94D/L96F/L98F/Q100R,N52S/N57Y/H94D/L96F/L98F/Q100R/G103E/F120S, N52H/F78L/Q100R,N52H/N57Y/Q100R/V110D, N52H/N57Y/R75Q/Q100R/V110D, N52H/N57Y/Q100R,N52H/N57Y/L74Q/Q100R/V110D, N52H/Q100R, N52H/S121G,A20V/N52H/N57Y/Q100R/S109G, N52H/N57Y/R61S/Q100R/V110D/L173S,N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S, N52H/N57Y, N52S/F120S,N52S/V97A, N52S/G72R, N52S/A71T/A117T, N52S/E220G,Y47H/N52S/V107A/F120S, N52H/N57Y/Q100R/V110D/S132F/M175T,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R,Q37R/N52H/N57Y/Q100R/V110N/S142F/C198R/D217V/R221G,N52H/N57Y/Q100R/V110D/C198R,N52H/N57Y/Q100R/V110D/V116A/L161M/F172S/S192G/C198R,F27S/N52H/N57Y/V110N, N52S/H94E/L96I/S109N/L166Q,S18R/N52S/F93L/I143V/R221G, A20T/N52D/Y146C/Q164L,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T,N52S/H94E/L96I/V122M, N52H/N57Y/H94E/L96I/F120I/S126T/W153R/I218N,M10V/S18R/N30D/N52S/S126R/T139S/L203F, S25G/N30D/N52S/F120S/N227K,N30D/N52S/L67P/Q100K/D217G/R221K/T225S,N52H/N57Y/Q100R/V110D/A117T/T190S/C198R,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R,N52H/N57Y/V110A/C198R/R221I,M10I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M,C198R,N52H/N57Y/R61C/Y62F/Q100R/V110N/F120S/C198R,N52H/N57Y/Q100R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/N144D/F172S/C198R, N52S/H94E/L98F/Q100R,N52S/E90A, N30D/K42E/N52S, N52S/F120S/I143V/I224V,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/S54P, T38P/N52S/N57D,E111del, Y33del, N52H/C140del/T225A, N52H/F78L/Q100R/C198R,N52H/N57Y/R75Q/Q100P/V110D, N52H/N57Y/L74Q/V110D/S192G,N52H/S121G/C198R, N52S/F120S/N227K, N52S/A71T/A117T/T190A/C198R,T43A/N52H/N57Y/L74Q/D89G/V110D/F172S, N52H/N57Y/Q100R/V110D/S132F/M175T,N52H/N57Y/Q100R/V1071/V110D/1154F/C198R/R221G, N84Q, N119Q, N168Q,N207Q, N52Q, N52Q/N207Q, N168Q/N207Q, N52Q/N168Q, N84Q/N207Q,N155Q/N207Q, N119Q/N168Q, N119Q/N207Q, N119Q/N155Q, N52Q/N84Q,N52Q/N119Q, N84Q/N119Q, N52Q/N84Q/N168Q, N52Q/N84Q/N207Q,N84Q/N155Q/N168Q, N84Q/N168Q/N207Q, N84Q/N155H/N207Q, N155Q/N168Q/N207Q,N119Q N155Q/N168Q, N119Q/N168Q/N207Q, N84Q/N119Q/N207Q,N119Q/N155H/N207Q, N84Q/N119Q/N155Q, N52Q/N119Q/N155Q, N52H/N84Q/N119Q,N52H/N84Q, N52H/N84Q/N168Q, N52H/N84Q/N207Q, N52H/N84Q/N168Q/N207Q,N52Q/N84Q/N155Q, N52Q/N84Q/N168Q, N52Q/N84Q/N155Q/N168Q,N52Q/N84Q/N119Q/N168Q, N84Q/N119Q/N155Q/N168Q, N84Q/N155Q/N168Q/N207Q,N84Q/N119Q/N155Q/N207Q, N52Q/N84Q/N119Q/N207Q, N52Q/N84Q/N119Q/N155Q,N52Q/N84Q/N119Q/N155Q/N207Q, N84Q/N119Q/N155Q/N168Q/N207Q, Q100R,F138L/L203P, N52Y/F138L/L203P, N57Y/Q100R/C198R, N57Y/F138L/L203P,Q100R/F138L, L203P, N52H/N57Y/Q100R/H115R/C198R,N52H/N57Y/Q100R/F172S/C198R, N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/1143V/F172S/C198R,N52H/N57Y/Q100R/L102R/H115R/F172S/C198R, N52H/V122A/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/N194D, N52H/N57Y/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/H115R, N52H/N57Y/Q100R/H115R,N52H/N57Y/Q100R/H115R/F172S/1224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/F172S, N52H/Q100R/H115R/1143T/F172S,N52H/N57Y/Q100P/H115R/F172S, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/E90A/H115R, N30D/K42E N52S/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N52S/H115R/F120S/I143V/C198R, N52S/H115R/F172S/C198R,N52H/N57Y/Q100P/C198R, N52H/N57Y/Q100P H115R/F172S/C198R,N52H/N57Y/Q100P/F172S/C198R, N52H/N57Y/Q100P/H115R,N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R, N52H/Q100R/H115R/F172S,N52H/Q100R/ F172S/C198R, N52H/Q100R/H115R/F172S/C198R, orN52H/N57Y/Q100R/F172S/C198R.

In some of any such embodiments, the one or more amino acidmodifications are at a position(s) corresponding to a position selectedfrom 52, 57, 100, 110, or 198. In some of any such embodiments, the oneor more amino acid modifications are selected from N52H, N52D, N52S,N52K, N52Q, S54A, S54P, N57Y, Q100P, Q100R, V110A, V110D, C198R, or aconservative amino acid substitution thereof. In some embodiments, thevariant ICOSL further includes one or more additional modification, suchas any as described herein. In some of any such embodiments, the variantICOSL polypeptide further includes one or more amino acid modificationsselected from V11E, E16V, N30D, K42E, N52H, N52S, N52Y, N57Y, E90A,H94E, L96I, L98F, Q100R, Q100P, L102R, V110A, V110D, H115R, F120S,V122A, F138L, I143V, V152C, K156M, K156R, F172S, N194D, C198R, L203P,V210A, S212G, I218T, R221I, I224V, or a conservative amino acidsubstitution thereof.

In some of any such embodiments, the one or more amino acidmodifications are N52H/N57Y/Q100R/C198R, N52H/N57Y/Q100R/V122A,N52H/N57Y/Q100R/F172S, N52Y/N57Y/F138L/L203P,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T, N52H/N57Y/Q100R,N52H/Q100R, N52H/N57Y/Q100R/V110D/C198R/S212G,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/V152C/K156M/C198R, N30D/K42E/N52S,N52S/F120S/I143V/I224V, N52S/E90A, N52H/N57Y/V110A/C198R/R221I,N52H/N57Y/Q100P, or N52S/N194D.

In some embodiments of any one of the variant ICOSL polypeptidesdescribed above, the variant ICOSL polypeptide includes the IgV domainor a specific fragment thereof and the IgC domain or a specific fragmentthereof. In some embodiments of any one of the variant ICOSLpolypeptides described above, the variant ICOSL polypeptide includes thesequence of amino acids set forth in any of SEQ ID NOS: 109-142, 239,280-325, 364-381, 387-424, 427-433, 435-470 or a specific bindingfragment thereof, or a sequence of amino acids that exhibits at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity to any of SEQ ID NOS: 109-142, 239, 280-325,364-381, 387-424, 427-433, 435-470 or a specific binding fragmentthereof and that contains the one or more of the amino acidsubstitutions.

In some embodiments of any one of the variant ICOSL polypeptidesdescribed above, the variant ICOSL polypeptide includes the IgV domainor a specific binding fragment thereof. In some embodiments, the IgVdomain or specific binding fragment thereof is the only ICOSL portion ofthe variant ICOSL polypeptide. In some embodiments, the IgC domain orspecific binding fragment thereof is the only ICOSL portion of thevariant ICOSL polypeptide.

In some embodiments of any one of the variant ICOSL polypeptidesdescribed above, the variant ICOSL polypeptide includes the sequence ofamino acids set forth in any of SEQ ID NOS: 197-199, 201-208, 210, 212,240, 326-340, 382-386, 425-426, and 434 or a specific binding fragmentthereof, a sequence of amino acids that exhibits at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to any of SEQ ID NOS: 197-199, 201-208, 210, 212, 240, 326-340,382-386, 425-426, and 434 or a specific binding fragment thereof andthat contains the one or more of the amino acid substitutions.

In some embodiments of any one of the variant ICOSL polypeptidesdescribed above, the variant ICOSL polypeptide specifically binds to theectodomain of ICOS, CD28, or CTLA-4 with increased affinity compared tothe unmodified ICOSL. In some embodiments, the variant ICOSL polypeptidespecifically binds to the ectodomain of ICOS or CD28 with increasedaffinity compared to the unmodified ICOSL. In some embodiments, thevariant ICOSL polypeptide specifically binds to the ectodomain of ICOSand the ectodomain of CD28 each with increased affinity compared to theunmodified ICOSL.

In some of any such embodiments, the variant ICOSL polypeptidespecifically binds to the ectodomain of CD28 with increased affinitycompared to the unmodified ICOSL. In some of any such embodiments, theincreased affinity to the ectodomain of CD28 is increased more than1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 60-foldcompared to the unmodified ICOSL. In some embodiments, the variant ICOSLpolypeptide specifically binds to the ectodomain of ICOS with increasedaffinity compared to the unmodified ICOSL. In some of any suchembodiments, the increased affinity to the ectodomain of ICOS isincreased more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 40-fold, 50-fold,60-fold or 70-fold compared to the unmodified ICOSL.

In some embodiments of any one of the variant ICOSL polypeptides, theone or more amino acid substitutions correspond to position(s) selectedfrom 52, 54 or 57. In some embodiments, the one or more amino acidmodifications are selected from N52H, N52D, N52Q, N52S, N52Y, N52K,S54A, S54P, N57D, N57Y or a conservative amino acid substitutionthereof. In some of any such embodiments, the one or more amino acidsubstitutions modifications correspond to position(s) selected from 52or 57. In some embodiments, the one or more amino acid substitutions areselected from N52H, N52D, N52S, N52K or N57Y. In some embodiments, anyone of the variant ICOSL polypeptides further includes one moreadditional amino acid modification, such as any as described. In someembodiments of any one of the variant ICOSL polypeptides describedabove, the variant ICOSL polypeptide further includes one or more aminoacid modifications selected from N52H, N52D, N52S, N52Y, N52K, S54A,S54P, N57Y, R75Q, L80P, K92R, S99G, H94D, L96F, L98F, L96I, S99G, Q100R,Q100P, G103E, T113E, F120S, H129P, S130G, Q133H, F138L, C140D, C140del,I143T, Y146C, V151A, Y152C, Y152H, D158G, L161P, C198R, N201S, L203P,L208P or T225A, or a conservative amino acid substitution thereof.

In some of any such embodiments, the one or more amino acidmodifications are selected from N52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P,N52S/Y146C/Y152C, N52H/C198R, N52H/C140del/T225A, N52H/C198R/T225A,N52H/K92R, N57Y/Q100P, N52S/C198R, N52Y/N57Y/Y152C,N52Y/N57Y/H129P/C198R, N52H/L161P/C198R, N52S/T113E, N52S/S54P,N52K/L208P, N52S/Y152H, N52H/I143T, N52S/R75Q/L203P, N52S/D158G,N52D/Q133H, N52H/ N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R,N52S/N194D, N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/S54P,T38P/N52S/N57D, N52H/C140del/T225A, N52H/F78L/Q100R/C198R,N52H/N57Y/R75Q/Q100P/V110D, N52H/N57Y/L74Q/V110D/S192G,N52H/S121G/C198R, N52S/F120S/N227K, N52S/A71T/A117T/T190A/C198R,T43A/N52H/N57Y/L74Q/D89G/V110D/F172S, N52H/N57Y/Q100R/V110D/S132F/M175T,N52H/N57Y/Q100R/V107I/V110D/I154F/C198R/R221G, N52Q/N207Q, N52Q/N168Q,N52Q/N84Q, N52Q/N119Q, N52Q/N84Q/N168Q, N52Q/N84Q/N207Q,N52Q/N119Q/N155Q, N52H/N84Q/N119Q, N52H/N84Q, N52H/N84Q/N168Q,N52H/N84Q/N207Q, N52H/N84Q/N168Q/N207Q, N52Q/N84Q/N155Q,N52Q/N84Q/N168Q, N52Q/N84Q/N155Q/N168Q, N52Q/N84Q/N119Q/N168Q,N52Q/N84Q/N119Q/N207Q, N52Q/N84Q/N119Q/N155Q,N52Q/N84Q/N119Q/N155Q/N207Q, N52Y/F138L/L203P, N57Y/Q100R/C198R,N57Y/F138L/L203P, N52H/N57Y/Q100R/H115R/C198R,N52H/N57Y/Q100R/F172S/C198R, N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R,N52H/N57Y/Q100R/L102R/H115R/F172S/C198R, N52H/V122A/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/N194D, N52H/N57Y/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/H115R, N52H/N57Y/Q100R/H115R,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/F172S, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/E90A/H115R, N30D/K42E/N52S/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N52S/H115R/F120S/I143V/C198R, N52S/H115R/F172S/C198R,N52H/N57Y/Q100P/C198R, N52H/N57Y/Q100P H115R/F172S/C198R,N52H/N57Y/Q100P/F172S/C198R, N52H/N57Y/Q100P/H115R,N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R, N52H/Q100R/H115R/F172S,N52H/Q100R/ F172S/C198R, N52H/Q100R/H115R/F172S/C198R, orN52H/N57Y/Q100R/F172S/C198R.

In some of any such embodiments, the one or more amino acidmodifications are selected from among N52Y/N57Y/F138L/L203P,N52H/N57Y/Q100P, N52S/Y146C/Y152C, N52H/C198R, N52H/C140del/T225A,N52H/C198R/T225A, N52H/K92R, N57Y/Q100P, N52S/C198R, N52Y/N57Y/Y152C,N52Y/N57Y/H129P/C198R, N52H/L161P/C198R, N52S/T113E, N52S/S54P,N52K/L208P, N52S/Y152H, N52H/I143T, N52S/R75Q/L203P, N52S/D158G, orN52D/Q133H. In some of any such embodiments, the one or more amino acidmodifications are selected from N52H/N57Y/F138L/L203P, N52H/N57Y/Q100P,N52H/K92R, N52H/C140del/T225A, N52H/C198R/T225A, N52H/K92R, N57Y/Q100P,N52Y/N57Y/H129P/C198R, N52H/L161P/C198R, N52K/L208P or N52H/I143T.

In some of any such embodiments, the variant ICOSL polypeptidespecifically binds to the ectodomain of CTLA-4 with increased affinitycompared to the unmodified ICOSL. In some aspects, the increasedaffinity to the ectodomain of CTLA-4 is increased more than 1.2-fold,1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 40-fold, 50-fold, 60-fold or 70-fold comparedto the unmodified ICOSL.

In some of any such embodiments, the variant polypeptide specificallybinds to the ectodomain of ICOS, CD28 or CTLA4 with increasedselectivity compared to the unmodified ICOSL. In some embodiments, theincreased selectivity includes a greater ratio of binding of the variantpolypeptide for one cognate binding partner selected from among ICOS,CD28 and CTLA4 versus another of the cognate binding partner compared tothe ratio of binding of the unmodified ICOSL polypeptide for the onecognate binding partner versus the another of the cognate bindingpartner. In some cases, the ratio is greater by at least or at leastabout 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold. 5-fold, 10-fold, 15-fold,20-fold, 30-fold, 40-fold, 50-fold or more.

In some embodiments of any one of the variant ICOSL polypeptides, thevariant ICOSL polypeptide further includes one or more amino acidselected from M10V, M10I, V11E, S13G, E16V, S18R, A20V, S25G, F27S,F27C, N30D, Q37R, K42E, T43A, Y47H, N52H, N52D, N52S, N52Q, N52Y, N52K,S54A, S54P, N57D, N57Y, R61S, R61C, Y62F, L67P, A71T, G72R, L74Q, R75Q,D77G, F78L, L80P, N84Q, D89G, E90A, K92R, F93L, H94E, H94D, L96F, L961,V97A, L98F, S99G, Q100R, Q100K, Q100P, L102R, G103E, V107A, V1071,S109G, S109N, V110D, V110N, V110A, T113E, H115R, H115Q, V116A, A117T,F120S, F1201, S121G, V122A, V122M, S126T, S126R, H129P, S130G,S132F,Q133H, E135K, F138L, T139S, C140D, C140del, S142F,I143V, I143T, N144D,Y146C, V151A, Y152C, Y152H,W153R, I154F, N155H, N155Q, K156M, D158G,L161P, L161M, L166Q, N168Q, F172S, L173S, M175T, T190A, T190S, S192G,V193M, N194D, C198R, N201S, L203P, L203F, N207Q, L208P, V210A, S212G,D217V, I218T, I218N, E220G, R221G, R221I, I224V, T225A, N227K, or aconservative amino acid substitution thereof. In some embodiments of anyone of the variant ICOSL polypeptides, the variant ICOSL polypeptidefurther includes one or more amino acid deletions corresponding toposition 140 of SEQ ID NO: 32.

In some embodiments of any one of the variant ICOSL polypeptides, theone or more amino acid substitutions are selected from amongN52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P, N52S/Y146C/Y152C, N52H/C198R,N52H/C140D/T225A, N52H/C198R/T225A, N52H/K92R, N57Y/Q100P, N52S/C198R,N52Y/N57Y/Y152C, N52Y/N57Y/H129P/C198R, N52H/L161P/C198R, N52S/T113E,N52D/S54P, N52K/L208P, N52S/Y152H, N52H/I143T, N52S/R75Q/L203P,N52S/D158G, N52D/Q133H, N52H/ N57Y/Q100R/V110D/C198R/S212G,N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/S54P, T38P/N52S/N57D,N52H/C140del/T225A, N52H/F78L/Q100R/C198R, N52H/N57Y/R75Q/Q100P/V110D,N52H/N57Y/L74Q/V110D/S192G, N52H/S121G/C198R, N52S/F120S/N227K,N52S/A71T/A117T/T190A/C198R, T43A/N52H/N57Y/L74Q/D89G/V110D/F172S,N52H/N57Y/Q100R/V110D/S132F/M175T, orN52H/N57Y/Q100R/V1071/V110D/1154F/C198R/R221G.

In some embodiments of any one of the variant ICOSL polypeptides, theone or more amino acid substitutions are selected fromN52H/N57Y/F138L/L203P, N52H/N57Y/Q100P, N52H/K92R, N52H/C140del/T225A,N52H/C198R/T225A, N52H/K92R, N57Y/Q100P, N52Y/N57Y/H129P/C198R,N52H/L161P/C198R, N52K/L208P, N52H/I143T, A20V/N52H/N57Y/Q100R/S109G,N52H/N57Y/R61S/Q100R/V110D/L173S,N52H/N57Y/Q100R/V1071/V110D/S132F/1154F/C198R/R221G,Q37R/N52H/N57Y/Q100R/V110N/S142F/C198R/D217V/R221G,N52H/N57Y/Q100R/V110D/C198R, F27S/N52H/N57Y/V110N,S18R/N52S/F93L/I143V/R221G, A20T/N52D/Y146C/Q164L,N52H/N57Y/H94E/L96I/F120I/S126T/W153R/I218N,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R, orM10I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M/C198R.

In some embodiments of any one of the variant ICOSL polypeptides, thevariant ICOSL polypeptide specifically binds to the ectodomain of ICOSor CD28 with increased affinity and specifically binds to the ectodomainof the other of ICOS or CD28 with decreased affinity compared to theunmodified ICOSL. In some embodiments, the variant ICOSL polypeptidespecifically binds to the ectodomain of ICOS with increased affinity andspecifically binds to the ectodomain of CD28 with decreased affinitycompared to the unmodified ICOSL. In some embodiments, the one or moreamino acid substitutions are selected from N57Y/Q100P, N52S/S130G/Y152C,N52S/Y152C, N52Y/N57Y/Y152C, N52H/L161P/C198R, N52H/L161P/C198R,N52S/L80P, A20V/N52H/N57Y/Q100R/S109G, N52H/N57Y/R61S/Q100R/V110D/L173S,N52H/N57Y/Q100R/V107I/V110D/S132F/I154F/C198R/R221G,Q37R/N52H/N57Y/Q100R/V110N/S142F/C198R/D217V/R221G,N52H/N57Y/Q100R/V110D/C198R, F27S/N52H/N57Y/V110N,S18R/N52S/F93L/I143V/R221G, A20T/N52D/Y146C/Q164L,N52H/N57Y/H94E/L96I/F120I/S126T/W153R/I218N,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R, orM10I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M/C198R.

In some embodiments, the variant ICOSL polypeptide specifically binds tothe ectodomain of CD28 with increased affinity and specifically binds tothe ectodomain of ICOS with decreased affinity compared to theunmodified ICOSL. In some embodiments, the variant ICOSL polypeptideincludes the amino acid substitutions N52S/R75Q/L203P or N30D/K42E/N52S.

In some embodiments of any one of the variant ICOSL polypeptides, theICOS is a human ICOS. In some embodiments, the CD28 is a human CD28.

In some embodiments of any one of the variant ICOSL polypeptides, thebinding is altered (increased or decreased) more than 1.2-fold,1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 30-fold 40-fold or 50-fold compared to theunmodified ICOSL.

In some embodiments of any one of the variant ICOSL polypeptides, thevariant ICOSL polypeptide is a soluble protein.

In some embodiments of any one of the variant ICOSL polypeptides, thevariant ICOSL polypeptide is linked to a multimerization domain. In someembodiments, the variant ICOSL polypeptide is a multimeric polypeptide,optionally a dimeric polypeptide, comprising a first variant ICOSLpolypeptide linked to a multimerization domain and a second variantICOSL polypeptide linked to a multimerization domain. In someembodiments, the first variant ICOSL polypeptide and the second variantICOSL polypeptide are the same or different. In some embodiments, themultimerization domain is an Fc domain or a variant thereof with reducedeffector function. In some embodiments of any one of the variant ICOSLpolypeptides, the variant ICOSL polypeptide is linked to a moiety thatincreases biological half-life of the polypeptide.

In some embodiments, the variant ICOSL polypeptide is linked to an Fcdomain or a variant thereof with reduced effector function. In someembodiments, the Fc domain is mammalian, optionally human; or thevariant Fc domain includes one or more amino acid modifications comparedto an unmodified Fc domain that is mammalian, optionally human. In someembodiments, the Fc domain or variant thereof includes the sequence ofamino acids set forth in SEQ ID NO:226 or SEQ ID NO:227 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO:226 or SEQ ID NO:227.

In some embodiments, the Fc domain includes one or more amino acidmodifications selected from among E233P, L234A, L234V, L235A, L235E,G236del, G237A, S267K, R292C, N297G and V302C, each by EU numbering. Insome aspects, the Fc domain includes the amino acid modification C220Sby EU numbering. In some embodiments, the Fc domain includes thesequence of amino acids set forth in any of SEQ ID NOS:474, 476, 477,478 or a sequence of amino acids that exhibits at least 85% sequenceidentity to any of SEQ ID NOS: 474, 476, 477, 478 and that contains theone or more amino acid modifications and/or exhibits reduced effectorfunction.

In some embodiments, the variant ICOSL polypeptide is linked indirectlyto the multimerization domain or Fc via a linker, optionally a G4Slinker.

In some embodiments of any one of the variant ICOSL polypeptides, thevariant ICOSL polypeptide is a transmembrane immunomodulatory proteinthat further contains a transmembrane domain linked to the extracellulardomain (ECD) or specific binding fragment thereof of the variant ICOSLpolypeptide. In some embodiments, the transmembrane domain includes thesequence of amino acids set forth as residues 257-277 of SEQ ID NO:5 ora functional variant thereof that exhibits at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to residues 257-277 of SEQ ID NO:5. In some embodiments, thevariant ICOSL polypeptide further includes a cytoplasmic signalingdomain linked to the transmembrane domain. In some embodiments, thecytoplasmic signaling domain includes the sequence of amino acids setforth as residues 278-302 of SEQ ID NO:5 or a functional variant thereofthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to residues 278-302 of SEQID NO:5.

In some of any such embodiments, the variant ICOSL polypeptidecomprising the sequence of amino acids set forth in any of SEQ IDNOS:494-503 or a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to any of SEQ ID NOS:494-503 and that comprises one ormore amino acid modifications as described.

In some embodiments of any one of the variant ICOSL polypeptides, theICOSL variant increases IFN-gamma (interferon-gamma) expression relativeto the unmodified ICOSL in an in vitro primary T-cell assay. In someembodiments of any one of the variant ICOSL polypeptides describedabove, the variant ICOSL decreases IFN-gamma (interferon-gamma)expression relative to the unmodified ICOSL in an in vitro primaryT-cell assay. In some embodiments of any one of the variant ICOSLpolypeptides described above, the variant ICOSL polypeptide isdeglycosylated.

In some embodiments, provided herein is an immunomodulatory proteincomprising the variant ICOSL polypeptide according to any one of theprovided embodiments linked to a second polypeptide comprising animmunoglobulin superfamily (IgSF) domain. In some embodiments, the IgSFdomain is affinity modified and exhibits altered binding to one or moreof its cognate binding partner(s) compared to the unmodified orwild-type IgSF domain. In some embodiments, the IgSF domain exhibitsincreased binding to one or more of its cognate binding partner(s)compared to the unmodified or wild-type IgSF domain. In someembodiments, the variant ICOSL polypeptide is a first variant ICOSLpolypeptide and the IgSF domain of the second polypeptide is an IgSFdomain from a second variant ICOSL polypeptide according to any one ofthe provided embodiments, wherein the first and second variant ICOSL arethe same or different. In some embodiments, the variant ICOSLpolypeptide is capable of specifically binding to CD28 or ICOS and theIgSF domain of the second polypeptide is capable of binding to a cognatebinding partner other than one specifically bound by the variant ICOSLpolypeptide. In some embodiments, the IgSF domain is from a member ofthe B7 family. In some embodiments, the IgSF domain is atumor-localizing moiety that binds to a ligand expressed on a tumor oris an inflammatory-localizing moiety that binds to a ligand expressed ona cell or tissue of an inflammatory environment. In some embodiments,the IgSF domain is a tumor-localizing moiety that binds to a ligandexpressed on a tumor. In some embodiments, the ligand is B7H6. In someembodiments, the IgSF domain is from NKp30. In some embodiments, theIgSF domain is or includes an IgV domain. In some embodiments, thevariant ICOSL polypeptide is or includes an IgV domain.

In some embodiments according to any one of the immunomodulatoryproteins, the immunomodulatory protein includes a multimerization domainlinked to one or both of the variant ICOSL polypeptide or the secondpolypeptide comprising the IgSF domain. In some embodiments, themultimerization domain is an Fc domain or a variant thereof with reducedeffector function. In some embodiments, the immunomodulatory protein isdimeric. In some embodiments, the immunomodulatory protein ishomodimeric. In some cases, the immunomodulatory protein isheterodimeric.

In some embodiments, provided herein is a conjugate comprising a variantICOSL polypeptide according to any one of the embodiments or animmunomodulatory polypeptide according to any one of the embodimentslinked to a moiety. In some embodiments, the moiety is a targetingmoiety that specifically binds to a molecule on the surface of a cell.In some embodiments, the targeting moiety specifically binds to amolecule on the surface of an immune cell. In some embodiments, theimmune cell is an antigen presenting cell or a lymphocyte. In someembodiments, the targeting moiety is a tumor-localizing moiety thatbinds to a molecule on the surface of a tumor. In some embodiments, themoiety is a protein, a peptide, nucleic acid, small molecule ornanoparticle. In some embodiments, the moiety is an antibody orantigen-binding fragment. In some embodiments, the conjugate isdivalent, tetravalent, hexavalent or octavalent.

In some embodiments, provided herein is a nucleic acid molecule encodinga variant ICOSL according to any one of the provided embodiments or animmunomodulatory polypeptide according to any one of the providedembodiments. In some embodiments, the nucleic acid molecule is asynthetic nucleic acid. In some embodiments, the nucleic acid is cDNA.

In some embodiments, provided herein is a vector comprising the nucleicacid of any one of the provided embodiments. In some embodiments, thevector is an expression vector. In some embodiments, the vector is amammalian expression vector or a viral vector.

In some embodiments, provided herein is a cell comprising the vectoraccording to any one of the provided embodiments. In some embodiments,the cell is a mammalian cell. In some embodiments, the cell is a humancell.

In some embodiments, provided herein is a method of producing apolypeptide or an immunomodulatory protein, comprising introducing thenucleic acid molecule according to any one of the provided embodimentsor vector according to any one of the provided embodiments into a hostcell under conditions to express the protein in the cell. In someembodiments, the method further includes isolating or purifying thevariant ICOSL polypeptide or immunomodulatory protein from the cell.

In some embodiments, provided herein is a method of engineering a cellexpressing a variant ICOSL polypeptide, comprising introducing a nucleicacid molecule encoding the variant ICOSL polypeptide or immunomodulatorypolypeptide according to any one of the provided embodiments into a hostcell under conditions in which the polypeptide is expressed in the cell.In some embodiments, provided herein is an engineered cell, expressingthe variant ICOSL polypeptide, immunomodulatory polypeptide, the nucleicacid molecule, or the vector according to any one of the providedembodiments.

In some embodiments, the variant ICOSL polypeptide or immunomodulatorypolypeptide includes a signal peptide. In some aspects, the variantICOSL polypeptide or immunomodulatory polypeptide does not contain atransmembrane domain and/or is not expressed on the surface of the cell.In some cases, the variant ICOSL polypeptide or immunomodulatorypolypeptide is secreted from the engineered cell. In some aspects, theengineered cell contains a variant ICOSL polypeptide that contains atransmembrane domain and/or is the transmembrane immunomodulatoryprotein according to any of the provided embodiments. In someembodiments, the variant ICOSL polypeptide is expressed on the surfaceof the cell.

In some embodiments, the engineered cell is an immune cell. In someembodiments, the immune cell is an antigen presenting cell (APC) or alymphocyte. In some embodiments, the engineered cell is a primary cell.In some embodiments, the engineered cell is a mammalian cell. In someembodiments, the engineered cell is a human cell. In some embodiments,the lymphocyte is a T cell. In some embodiments, the APC is anartificial APC. In some embodiments, the engineered cell furthercontains a chimeric antigen receptor (CAR) or an engineered T-cellreceptor (TCR).

Also provided is an infectious agent, comprising a nucleic acid moleculeencoding a variant ICOSL polypeptide or an immunomodulatory polypeptideaccording to any of the provided embodiments. In some embodiments, theencoded variant ICOSL polypeptide or immunomodulatory polypeptide doesnot contain a transmembrane domain and/or is not expressed on thesurface of a cell in which it is expressed. In some cases, the encodedvariant ICOSL polypeptide or immunomodulatory polypeptide is secretedfrom a cell in which it is expressed. In some cases, the encoded variantICOSL polypeptide contains a transmembrane domain. In some embodiments,the encoded variant ICOSL polypeptide is expressed on the surface of acell in which it is expressed.

In some of any such embodiments, the infectious agent is a bacteria or avirus. In some examples, the virus is an oncolytic virus. In someaspects, the oncolytic virus is an adenoviruses, adeno-associatedviruses, herpes viruses, Herpes Simplex Virus, Vesticular Stomaticvirus, Reovirus, Newcastle Disease virus, parvovirus, measles virus,vesticular stomatitis virus (VSV), Coxsackie virus or a Vaccinia virus.In some embodiments, the virus specifically targets dendritic cells(DCs) and/or is dendritic cell-tropic. In some embodiments, the virus isa lentiviral vector that is pseudotyped with a modified Sindbis virusenvelope product.

In some of any such embodiments, the infectious agent further comprisesa nucleic acid molecule encoding a further gene product that results indeath of a target cell or that can augment or boost an immune response.In some embodiments, the further gene product is selected from ananticancer agent, anti-metastatic agent, an antiangiogenic agent, animmunomodulatory molecule, an immune checkpoint inhibitor, an antibody,a cytokine, a growth factor, an antigen, a cytotoxic gene product, apro-apoptotic gene product, an anti-apoptotic gene product, a cellmatrix degradative gene, genes for tissue regeneration or areprogramming human somatic cells to pluripotency.

In some embodiments, provided herein is a pharmaceutical composition,comprising the variant ICOSL polypeptide according to any one of theprovided embodiments, an immunomodulatory protein according to any oneof the provided embodiments, a conjugate according to any one of theprovided embodiments, an engineered cell according to any one of theprovided embodiments, or an infectious agent according to any one of theprovided embodiments. In some embodiments, the pharmaceuticalcomposition further includes a pharmaceutically acceptable excipient. Insome embodiments, the pharmaceutical composition is sterile.

In some embodiments, provided herein is an article of manufactureincludes any of the provided pharmaceutical compositions in a vial. Insome embodiments, the vial is sealed. In some embodiments, also providedis a kit including any of the pharmaceutical compositions andinstructions for use. In some aspects, provided is a kit including thearticle of manufacture and instructions for use.

In some embodiments, provided herein is a method of modulating an immuneresponse in a subject, comprising administering the pharmaceuticalcomposition according to any one of the provided embodiments to thesubject. In some embodiments, the method includes administering theengineered cells as provided according to any one of the providedembodiments. In some embodiments, the engineered cells are autologous tothe subject. In some embodiments, the engineered cells are allogenic tothe subject. In some embodiments, modulating the immune response treatsa disease or condition in the subject.

In some embodiments, the immune response is increased. In someembodiments, an immunomodulatory protein or conjugate comprising avariant ICOSL polypeptide linked to a tumor-localizing moiety isadministered to the subject. In some cases, the tumor-localizing moietyis or includes a binding molecule that recognizes a tumor antigen. Insome aspects, the binding molecule includes an antibody or anantigen-binding fragment thereof or includes a wild-type IgSF domain orvariant thereof.

In some of any such embodiments, a pharmaceutical composition comprisingthe immunomodulatory protein according to any one of the providedembodiments or the conjugate according to any one of the providedembodiments is administered to the subject. In some embodiments, anengineered cell comprising a variant ICOSL polypeptide that is atransmembrane immunomodulatory protein is administered to the subjectand/or the engineered cell according to any one of the embodimentsdescribed above.

In some embodiments, an infectious agent encoding a variant ICOSLpolypeptide that is a transmembrane immunomodulatory protein isadministered to the subject, optionally under conditions in which theinfectious agent infects a tumor cell or immune cell and thetransmembrane immunomodulatory protein is expressed on the surface ofthe infected cell. In some aspects, the transmembrane immunomodulatoryprotein is any provided according to any one of the providedembodiments.

In some embodiments, the disease or condition is a tumor or cancer. Insome embodiments, the disease or condition is selected from melanoma,lung cancer, bladder cancer, a hematological malignancy, liver cancer,brain cancer, renal cancer, breast cancer, pancreatic cancer, colorectalcancer, spleen cancer, prostate cancer, testicular cancer, ovariancancer, uterine cancer, gastric carcinoma, a musculoskeletal cancer, ahead and neck cancer, a gastrointestinal cancer, a germ cell cancer, oran endocrine and neuroendocrine cancer.

In some embodiments, the immune response is decreased by the providedmethods of modulating the immune response. In some embodiments, avariant ICOSL polypeptide or immunomodulatory protein that is soluble isadministered to the subject. In some cases, the soluble polypeptide orimmunomodulatory protein is an Fc fusion protein. In some embodiments, apharmaceutical composition comprising a variant ICOSL polypeptideaccording to any one of the provided embodiments, or theimmunomodulatory protein according to any one of the providedembodiments is administered to the subject.

In some of any such embodiments, an engineered cell comprising asecretable variant ICOSL polypeptide is administered to the subject. Insome embodiments, an engineered cell according to any one of theprovided embodiments is administered to the subject. In someembodiments, an infectious agent encoding a variant ICOSL polypeptidethat is a secretable immunomodulatory protein is administered to thesubject, optionally under conditions in which the infectious agentinfects a tumor cell or immune cell and the secretable immunomodulatoryprotein is secreted from the infected cell.

In some embodiments, the disease or condition is an inflammatory orautoimmune disease or condition. In some embodiments, the disease orcondition is an Antineutrophil cytoplasmic antibodies (ANCA)-associatedvasculitis, a vasculitis, an autoimmune skin disease, transplantation, aRheumatic disease, an inflammatory gastrointestinal disease, aninflammatory eye disease, an inflammatory neurological disease, aninflammatory pulmonary disease, an inflammatory endocrine disease, or anautoimmune hematological disease. In some embodiments, the disease orcondition is selected from inflammatory bowel disease, transplant,Crohn's disease, ulcerative colitis, multiple sclerosis, asthma,rheumatoid arthritis, or psoriasis.

In some of any such embodiments of the variant ICOSL polypeptide, theamino acid modification is an amino acid substitution, insertion or adeletion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts impedance results reflecting cytotoxic killing activityof cells engineered with an anti-CD19 chimeric antigen receptor (CAR)alone or with an exemplary transmembrane immunomodulatory TIP (CD80-TIPor ICOSL-TIP) or the corresponding CD80 or ICOSL wild-type transmembraneprotein following co-culture with target antigen-expressing cells.Impedance was assessed using the Acea Real-Time Cell Analyzer (RTCA),which measures the impedance variations in the culture media of a96-well microelectronic plate (E-plate).

FIG. 2A depicts that primary T cells are effectively transduced withviruses encoding both the CAR and TIP proteins. Primary human T cellsactivated 48 hours with anti-CD3 plus anti-CD28 beads and were thentranduced with a Lenti-virus encoding an anti-CD19 CAR with a BFPreporter, plus a second Lenti-virus encoding and ICOSL TIP with a GFPreporter. The FACs plot shows BFP expression on the y-axis and GFPexpression on the x-axis and the percentage of T cells that fall intoeach quadrant are indicated. Results show that the cultures include CARonly transduced cells (upper left quadrant), TIP only transduced cells(lower right quadrant), cells transduced with both viruses (upper rightquadrant) and cells that were not transduced with either (lower left).In FIG. 2B, TIPs expressed on CAR-T cells provide co-stimulation to theCAR-T cells. CAR-T cells with or without TIP co-transduction werelabeled with Cell-Trace Far Red and incubated with the CD19+ NALM6 cellline to engage the CAR. Proliferation was assessed by the percentage ofCAR-expressing cells that had diluted out the fluorescent dye. Cellstransduced with mutated TIPs showed an increase proliferation of CAR+ Tcells compared to those without TIPs or those transduced with wild-typeICOSL. Mock transduced cells that lacked CAR expression failed toproliferate in this assay.

FIG. 3A-3B demonstrate, via cytokine release, the costimulatory capacityof wild-type (WT) or variant ICOSL when coimmobilized with anti-CD3. 10nM anti-CD3 was wet coated to the wells of 96-well flat bottomedpolystyrene tissue culture plates with 40 nM (arrows) or 10 nM WT orvariant ICOSL. 100,000 purified CD4⁺ and CD8⁺ (pan) T-cells cells wereadded and supernatant was harvested 72 hours later for ELISA analysisfor cytokine release. FIG. 3A shows IFN-gamma and FIG. 3B shows IL-17protein levels secreted from pan T-cells. Graphs are representative oftypical IFN-gamma and IL-17 responses from pan T-cell costimulation.

FIG. 4A-4B demonstrate, via proliferation, the costimulatory capacity ofwild-type (WT) or variant ICOSL when coimmobilized with anti-CD3.CFSE-labeled pan T-cells were incubated in anti-CD3 and ICOSL coatedplates as previously described for 72 hours. Cells were harvested,washed, stained with fluorescently conjugated anti-CD4 or anti-CD8antibodies, and analyzed by flow cytometry. Gates and cytometer voltageswere set using non-stimulated control CFSE-labeled T-cells.Proliferation was determined by CFSE dilution from control. FIG. 4Ashows percent of total proliferating (arrows), CD4⁺ (solid bar), andCD8⁺ cells (hatched bar) T-cells following 40 nM ICOSL costimulation.FIG. 4B shows percent of total pan T-cell proliferation following 10 nMICOSL costimulation. Graphs are representative of typical proliferativeresponse from pan T-cell costimulation.

FIG. 5 depicts ICOSL vIgD candidate function in a humanMixed-Lymphocyte-Reation (MLR). ICOSL variants and their mutations arelisted on the x-axis, along with wild-type ICOSL, negative controlsPDL2-Fc and human IgG, as well as the positive control benchmarkmolecule CTLA-Ig Belatacept. The line across the graph represents thebaseline amount of IFN-gamma detected in the supernatants of negativecontrol cultures. For each ICOSL variant candidate or control, threedifferent concentrations were tested with arrows indicating the highestconcentration of protein in cultures at 40 nM. The majority of ICOSLvariant candidates show superior antagonistic activity at all threeconcentrations tested compared to belatacept as reflected by the lowerconcentration of IFN-gamma in those cultures.

FIG. 6 depicts the inhibition of soluble ICOSL Fc-fusion proteins on Band T cell responses in a B-T co-culture assay. FIG. 6A depicts solubleICOSL Fc-fusion proteins inhibition of T cell-driven B cellproliferation. Purified CD4+ T cells and B cells from a single donorwere CFSE-labeled and co-incubated at a 1:1 ration in the presence orabsence of the indicated mitogens with or without the indicated ICOSLFc-fusion proteins. Cells were stimulated with Staph enterotoxin B (SEB)at 100 ng/ml, Pokeweed mitogen (PWM) at 1 mg/ml, or both. ICOSLFc-fusion proteins were included at a final concentration of 40 nM andcultures were incubated for 7 days and subjected to FACS analysis. Thenumber of divided B cells was determined from the number of cells in thecultures that had diluted their CFSE. All of the ICOSL Fc-fusionproteins tested except for wild-type reduced B cell proliferation. FIG.6B-D show ICOSL Fc-fusion proteins inhibited cytokine T cell cytokineproduction in B-T co-cultures. Supernatants from the cultures describedabove were harvested on day 7 and analyzed for cytokine content using aLEGENDplex Human Th Cytokine Panel (Biolegend). T cell production ofIL-5 (FIG. 6B), IL-13 (FIG. 6C) and IL-21 (FIG. 6D) is attenuated byinclusion of ICOSL Fc-fusion proteins.

FIG. 7A-7D depicts different endpoints in a mouse model of Graft VerseHost Disease (GVHD) where human PBMC cells were adoptively transferredinto immunodefecient NSG murine hosts. FIG. 7A shows survival curves ofthe treated animals. Aggressive disease course and subsequent mortalitywas observed in the saline control animals, with similar survivalobserved in the animals treated with wild-type ICOSL-Fc, as well as theN52H/I143T ICOSL variant. Variant N52H/N57Y/Q100P had improved survivalrates comparable to the clinical benchmark belatacept. FIG. 7B showssimilar trends in body weight loss, with ICOSL variant N52H/N57Y/Q100Pdemonstrating similar weight maintenance as animals treated withbelatacept, even though all other groups experienced rapid weight loss.FIG. 7C shows clinical scores from standardized GVHD Disease ActivityIndex (DAI) observations, again showing lower scores in animals treatedwith the ICOSL variant N52H/N57Y/Q100P that are comparable to theclinical benchmark belatacept while the other groups of animalsexperienced higher DAI scores. FIG. 7D depicts a flow cytometricmeasurement of CD4 and CD8 percentages in blood from experimentalanimals measured on day 14. The percentage of CD8 cells betweenexperimental groups was largely the same, however, animals treated withICOSL variant N52H/N57Y/Q100P and belatacept have lower percentages ofCD4 cells compared to the other experimental groups.

FIG. 8 shows localized costimulatory activity conveyed by the indicatedvariant stack molecule vIgD C-L, where C represents an ICOSLcostimulatory domain and L represents a NKp30 localizing domain. In thisassay, target K562 cells expressing the localizing surface protein,B7-H6, were cultured in the presence of anti-CD3 with human T cells andT cell activation was assessed by IFN-gamma levels in culturesupernatants. Including anti-CD3 alone or no stack variant Fc moleculesdid not induce T cell activation. Similarly, cells cultured with onlythe wild-type localizing NKp30 domain alone or the wild-typecostimulatory ICOSL domain alone as Fc fusion proteins did not result inT cell activation. A stacked domain containing the wild-type version ofboth the costimulatory domain and localizing domain induced measurableIFN-gamma at the highest concentration tested, however, the variantlocalizing costimulatory stack induced greater than two fold higherIFN-gamma levels at the highest concentration, and IFN-gamma levels thatwere still observed as the concentrations were titrated down.

FIG. 9 summarizes changes in ear thickness in mice from a standard modelof Delayed-Type Hypersensitivity (DTH). PBS treated animals sensitizedwith ovalbumin and subsequently challenged in the ear with the sameprotein, show the highest level of measured ear swelling. Mice treatedwith clinical benchmark Abatacept have slightly reduced ear swellingfollowing ear challenge. All five ICOSL variant treatment groupsdemonstrated equal or improved reductions in ear swelling compared toAbatacept.

FIG. 10A-C depicts various exemplary configurations of a variant IgSFdomain (vIgD) conjugated to an antibody (V-Mab). FIG. 10A shows variousconfigurations in which a vIgD is linked, directly or indirectly, to theN- and/or C-terminus of the light chain of an antibody. FIG. 10B showsvarious configurations in which a vIgD is linked, directly orindirectly, to the N- and/or C-terminus of the heavy chain of anantibody. FIG. 10C depicts the resulting V-Mab configurations when alight chain of FIG. 10A and a heavy chain of FIG. 10B are co-expressedin a cell.

FIG. 11A-11B demonstrate V-Mab specificity for cognate binding partners.Binding assays were performed on Expi293 cells transiently transfectedwith DNA for mammalian surface expression of human HER2, CD28, CTLA-4,or ICOS. 200,000 transfected cells were incubated with 100,000 pM to 100pM parental antibody (C1) or various V-Mabs (C2-9). Unbound antibody wasremoved, bound antibody detected with fluorescently conjugatedanti-human IgG, and the cells were analyzed by flow cytometry for MFIand percentage positive based on Fc controls. FIG. 11A shows binding ofthe V-Mabs to HER2 transfectants at levels similar to the parentalantibody. Binding to mock transfected cells is observed with all V-Mabs,though not WT ICOSL, due to low levels of endogenous HER2 expression onExpi293 parental cells. FIG. 11B shows binding of the parentalIgSF-domain (N52H/N57Y/Q100P) to its cognate partners is maintained orincreased (C2, C3, C4, C5, C6, C8, C9) by V-Mabs.

FIG. 12 demonstrates V-Mab costimulatory and proliferative capacity whencoimmobilized with anti-CD3. 10 nM anti-CD3 was wet coated to the wellsof 96-well flat bottomed polystyrene tissue culture plates with 30 nM to3 nM parental antibody, V-Mabs, or Fc controls. CFSE-labeled pan T-cellswere added for 72 hours. IFN-gamma secretion was measured by ELISA andtotal T-cell proliferation was measured by flow cytometric analysis ofCFSE-dilution. IFN-gamma secretion and proliferation of IgSF-domain(N52H/N57Y/Q100P) is greater than WT ICOSL. V-Mabs demonstrate increasedcytokine and proliferative costimulatory capacity over the parentalIgSF.

FIG. 13A-13C depicts various formats of the provided variant IgSF domainmolecules. FIG. 13A depicts soluble molecules, including: (1) a variantIgSF domain (vIgD) fused to an Fc chain; (2) a stack molecule containinga first variant IgSF domain (first vIgD) and a second IgSF domain, suchas a second variant IgSF domain (second vIgD); (3) a tumor targetingIgSF molecule containing a first variant IgSF domain (vIgD) and an IgSFdomain that targets to a tumor antigen, such as an NkP30 IgSF domain;and (4) a variant IgSF domain (vIgD) linked to an antibody (V-Mab). FIG.13B depicts a transmembrane immunomodulatory protein (TIP) containing avariant IgSF domain (vIgD), e.g. variant ICOSL, expressed on the surfaceof a cell. In an exemplary embodiment, the cognate binding partner ofthe transmembrane bound vIgD is a costimulatory receptor, e.g. CD28, andthe TIP containing the vIgD (e.g. ICOSL vIgD) agonizes the costimulatoryreceptor such that the TIP induces a positive signal in the cellexpressing the costimulatory receptor. FIG. 13C depicts a secretedimmunomodulatory protein (SIP) in which a variant IgSF domain (vIgD),e.g. variant ICOSL, is secreted from a cell, such as a first T cell(e.g. CAR T cell). In an exemplary embodiment, the cognate bindingpartner of the secreted vIgD is an activating receptor, e.g. CD28, whichcan be expressed on the first cell (e.g. T cell) and/or on a second cell(e.g. T cell; either endogenous or engineered, such as a CAR T cell).Upon binding of the SIP with its cognate binding partner, signaling viathe activating receptor is blocked. In all cases, the vIgD can be aV-domain (IgV) only, the combination of the V-domain (IgV) and C-domain(IgC), including the entire extracellular domain (ECD), or anycombination of Ig domains of the IgSF superfamily member.

FIG. 14 depicts an exemplary schematic of the activity of a variant IgSFdomain (vIgD) fused to an Fc (vIgD-Fc) in which the vIgD is a variant ofan IgSF domain of ICOSL. As shown, a soluble vIgD of ICOSL interactswith its cognate binding partners to block interactions of CD80(B7-1)/CD86 (B7-2) or ICOSL with CD28 or ICOS, respectively, therebyblocking costimulation by the CD28 and/or ICOS costimulatory receptors.

FIG. 15 depicts an exemplary schematic of a stack molecule forlocalizing the variant IgSF domain (vIgD) to a tumor cell. In thisformat, the stack molecule contains a first variant IgSF domain (firstvIgD) and a second IgSF domain (e.g. a second vIgD) in which the secondIgSF domain (e.g. a second vIgD) is a tumor-targeted IgSF domain thatbinds to a tumor antigen. An exemplary tumor-targeted IgSF domain is anIgSF domain of NkP30, which binds to the tumor antigen B7-H6. In thisdepiction, the vIgD is a variant of an IgSF domain of ICOSL. As shown,binding of tumor-targeted IgSF domain to the surface of the tumor celllocalizes the first vIgD on the tumor cell surface where it can interactwith one or more of its cognate binding partners (e.g. CD28 or ICOS)expressed on the surface of an adjacent immune cell (e.g. T cell) tostimulate the costimulatory receptor.

FIG. 16 depicts various exemplary configurations of a stack moleculecontaining a first variant IgSF domain (first vIgD), e.g. variant ICOSL,and a second IgSF domain, such as a second variant IgSF domain (secondvIgD). As shown, the first vIgD and second IgSF domain are independentlylinked, directly or indirectly, to the N- or C-terminus of an Fcsubunit. For generating a homodimeric Fc molecule, the Fc subunit is onethat is capable of forming a homodimer with a matched Fc subunit byco-expression of the individual Fc subunits in a cell. For generating aheterodimeric Fc molecule, the individual Fc subunits contain mutations(e.g. “knob-into-hole” mutations in the CH3 domain), such that formationof the heterodimer is favored compared to homodimers when the individualFc subunits are co-expressed in a cell.

FIG. 17 depicts an exemplary schematic of the activity of a variant IgSFdomain (vIgD) conjugated to an antibody (V-Mab) in which the antibody(e.g. anti-HER2 antibody) binds to an antigen on the surface of thetumor cell. In this depiction, the vIgD is a variant of an IgSF domainof ICOSL. As shown, binding of the antibody to the surface of the tumorcell localizes the vIgD on the tumor cell surface where it can interactwith one or more of its cognate binding partners expressed on thesurface of an adjacent immune cell (e.g. T cell) to agonize receptorsignaling. In an exemplary embodiment as shown, the variant IgSF domain(vIgD) is a variant of an IgSF domain of ICOSL. Binding of the ICOSLvIgD to CD28 or ICOS costimulatory receptors provides an agonist orcostimulatory signal.

FIG. 18 depicts the Nanostring transcriptional signature of primaryhuman T cells when incubated 10 nM anti-CD3 with 40 nM of an Fc-controlprotein, wild-type ICOSL-Fc, wild-type CD80-Fc, both of these proteins,or a variant ICOSLFc-fusion proteins with mutations as indicated. TotalRNA from samples was prepared from harvested cells and the RNA wastransferred to Nanostring and a Cancer Immune chip was used toquantitate transcripts of 750 gene in each sample. Altered transcriptsinclude those whose level is above or below the diagnol, including thenoted transcripts.

FIG. 19 depicts transcript levels of exemplary transcripts uponincubation as described in FIG. 18 for the indicated times in thepresence of the various immunomodulatory proteins.

FIG. 20A-B demonstrates VmAb mediated T-cell proliferation whenco-cultured with HER2 expressing targets. CFSE-labeled pan T-cells wereactivated with K562-derived artificial target cells displaying cellsurface anti-CD3 single chain Fv (OKT3) and HER2 in the presence ofVmAbs or control proteins. Proliferation was measured by flow cytometricanalysis of CFSE-dilution on CD4⁺ (left panel) or CD8⁺ (right panel)stained T-cells. In FIG. 20A, K562 cells were titrated and plated withT-cells for an effector:target (E:T) ratio of 40 to 1280:1. VmAbs,parental IgSF domain, or WT ICOSL were added at 1000 μM. In FIG. 20B,K562 cells were added to T-cells for an E:T ratio of 160:1. VmAbs orcontrol proteins were titrated and added at 3000 to 37 μM.

DETAILED DESCRIPTION

Provided herein are immunomodulatory proteins that are or comprisevariants or mutants of ICOS ligand (ICOSL) or specific binding fragmentsthereof that exhibit activity to bind to at least one target ligandcognate binding partner (also called counter-structure protein). In someembodiments, the variant ICOSL polypeptides contain one or more aminoacid modifications (e.g. amino acid substitutions, deletions oradditions) compared to an unmodified or wild-type ICOSL polypeptide. Insome embodiments, the one or more amino acid modifications (e.g. aminoacid substitutions, deletions or additions) are in an IgSF domain (e.g.IgV) of an unmodified or wild-type ICOSL polypeptide. In someembodiments, the variant ICOSL polypeptide exhibits altered, such asincreased or decreased, binding activity or affinity for at least onecognate binding partner, such as at least one of ICOS, CD28, or CTLA-4.In some embodiments, the immunomodulatory proteins are soluble. In someembodiments, the immunomodulatory proteins are transmembraneimmunomodulatory proteins capable of being expressed on the surface ofcells. In some embodiments, also provided herein are one or more otherimmunomodulatory proteins that are conjugates or fusions containing avariant ICOSL polypeptide provided herein and one or more other moietyor polypeptide.

In some embodiments, the variant ICOSL polypeptides and immunomodulatoryproteins modulate an immunological immune response, such as an increasedor decreased immune response. In some embodiments, the variant ICOSLpolypeptides and immunomodulatory proteins provided herein can be usedfor the treatment of diseases or conditions that are associated with adysregulated immune response.

In some embodiments, the provided variant ICOSL polypeptides modulate Tcell activation via interactions with costimulatory signaling molecules.In general, antigen specific T-cell activation requires two distinctsignals. The first signal is provided by the interaction of the T-cellreceptor (TCR) with major histocompatibility complex (MHC) associatedantigens present on antigen presenting cells (APCs). The second signalis costimulatory to TCR engagement and necessary to avoid T-cellapoptosis or anergy.

In some embodiments, under normal physiological conditions, the Tcell-mediated immune response is initiated by antigen recognition by theT cell receptor (TCR) and is regulated by a balance of co-stimulatoryand inhibitory signals (e.g., immune checkpoint receptors). The immunesystem relies on immune checkpoint receptors to prevent autoimmunity(i.e., self-tolerance) and to protect tissues from excessive damageduring an immune response, for example during an attack against apathogenic infection. In some cases, however, these immunomodulatoryproteins can be dysregulated in diseases and conditions, includingtumors, as a mechanism for evading the immune system.

In some embodiments, among known T-cell costimulatory receptors is CD28,which is the T-cell costimulatory receptor for the ligands B7-1 (CD80)and B7-2 (CD86) both of which are present on APCs. These same ligandscan also bind to the inhibitory T-cell receptor CTLA4 (cytotoxicT-lymphocyte-associated protein 4) with greater affinity than for CD28;the binding to CTLA-4 acts to down-modulate the immune response. ICOS(inducible costimulator) is another T-cell costimulatory receptor whichbinds to ICOS ligand (ICOSL) on APCs. In some cases, CD28 and CTLA-4also are known to interact with ICOSL at a binding site that overlapswith the binding of ICOSL to the T-cell costimulatory receptor ICOS (Yaoet al. (2011) Immunity, 34:729-740). Although CD28 and ICOS are relatedCD28 family activating receptors and share some intracellular signalingmotifs, costimulatory effects between CD28 and ICOS differ. For example,CD28 is expressed on both unactivated and activated T cells and itssignaling is important for IL-2 production and subsequent T celleffector function. ICOS is generally not expressed on the surface of Tcells until after T cell activation, and signaling through ICOS onactivated T cells supports specialized T cell subset differentiation.Thus, in some cases, costimulation by CD28 and ICOS yields overlappingand complementary effects.

In some embodiments, the T-cell costimulatory receptors CD28 and ICOShave distinct but complementary roles in modulating an immune response.Enhancement or suppression of the activity of these receptors hasclinical significance for treatment of inflammatory and autoimmunedisorders, cancer, and viral infections. In some cases, however,therapies to intervene and alter the costimulatory effects of bothreceptors are constrained by the spatial orientation requirements aswell as size limitations imposed by the confines of the immunologicalsynapse. In some aspects, existing therapeutic drugs, including antibodydrugs, may not be able to interact simultaneously with the multipletarget proteins involved in modulating these interactions. In addition,in some cases, existing therapeutic drugs may only have the ability toantagonize but not agonize an immune response. Additionally,pharmacokinetic differences between drugs that independently target oneor the other of these two receptors can create difficulties in properlymaintaining a desired blood concentration of such drug combinationsthroughout the course of treatment.

In some embodiments, the provided variant ICOSL polypeptides orimmunomodulatory proteins modulate (e.g. increase or decrease)immunological activity induced by costimulatory receptors CD28 or ICOS.Thus, in some embodiments, the provided polypeptides overcome theseconstraints by providing variant ICOSL (inducible costimulator ligand)with altered (e.g. increased or decreased) binding affinities to bothCD28 and ICOS, and, in some cases, CTLA-4, thereby agonizing orantagonizing the complementary effects of costimulation by receptors.Methods of making and using these variant ICOSL are also provided.

All publications, including patents, patent applications scientificarticles and databases, mentioned in this specification are hereinincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, including patent, patentapplication, scientific article or database, were specifically andindividually indicated to be incorporated by reference. If a definitionset forth herein is contrary to or otherwise inconsistent with adefinition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth herein prevails over the definitionthat is incorporated herein by reference.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

I. DEFINITIONS

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood by one of ordinaryskill in the art to which the claimed subject matter pertains. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.

The terms used throughout this specification are defined as followsunless otherwise limited in specific instances. As used in thespecification and the appended claims, the singular forms “a,” “an,” and“the” include plural referents unless the context clearly dictatesotherwise. Unless defined otherwise, all technical and scientific terms,acronyms, and abbreviations used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which theinvention pertains. Unless indicated otherwise, abbreviations andsymbols for chemical and biochemical names is per IUPAC-IUBnomenclature. Unless indicated otherwise, all numerical ranges areinclusive of the values defining the range as well as all integer valuesin-between.

The term “affinity modified” as used in the context of an immunoglobulinsuperfamily domain, means a mammalian immunoglobulin superfamily (IgSF)domain having an altered amino acid sequence (relative to thecorresponding wild-type parental or unmodified IgSF domain) such that ithas an increased or decreased binding affinity or avidity to at leastone of its cognate binding partners (alternatively “counter-structures”)compared to the parental wild-type or unmodified (i.e., non-affinitymodified) IgSF control domain. Included in this context is an affinitymodified ICOSL IgSF domain. In some embodiments, the affinity-modifiedIgSF domain can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or moreamino acid differences, such as amino acid substitutions, in a wildtypeor unmodified IgSF domain. An increase or decrease in binding affinityor avidity can be determined using well known binding assays such asflow cytometry. Larsen et al., American Journal of Transplantation, Vol5: 443-453 (2005). See also, Linsley et al., Immunity, 1: 7930801(1994). An increase in a protein's binding affinity or avidity to itscognate binding partner(s) is to a value at least 10% greater than thatof the wild-type IgSF domain control and in some embodiments, at least20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000%, or 10000%greater than that of the wild-type IgSF domain control value. A decreasein a protein's binding affinity or avidity to at least one of itscognate binding partner is to a value no greater than 90% of the controlbut no less than 10% of the wild-type IgSF domain control value, and insome embodiments no greater than 80%, 70% 60%, 50%, 40%, 30%, or 20% butno less than 10% of the wild-type IgSF domain control value. Anaffinity-modified protein is altered in primary amino acid sequence bysubstitution, addition, or deletion of amino acid residues. The term“affinity modified IgSF domain” is not be construed as imposing anycondition for any particular starting composition or method by which theaffinity-modified IgSF domain was created. Thus, the affinity modifiedIgSF domains of the present invention are not limited to wild type IgFdomains that are then transformed to an affinity modified IgSF domain byany particular process of affinity modification. An affinity modifiedIgSF domain polypeptide can, for example, be generated starting fromwild type mammalian IgSF domain sequence information, then modeled insilico for binding to its cognate binding partner, and finallyrecombinantly or chemically synthesized to yield the affinity modifiedIgSF domain composition of matter. In but one alternative example, anaffinity modified IgSF domain can be created by site-directedmutagenesis of a wild-type IgSF domain. Thus, affinity modified IgSFdomain denotes a product and not necessarily a product produced by anygiven process. A variety of techniques including recombinant methods,chemical synthesis, or combinations thereof, may be employed.

The term “allogeneic” as used herein means a cell or tissue that isremoved from one organism and then infused or adoptively transferredinto a genetically dissimilar organism of the same species. In someembodiments of the invention, the species is murine or human.

The term “autologous” as used herein means a cell or tissue that isremoved from the same organism to which it is later infused oradoptively transferred. An autologous cell or tissue can be altered by,for example, recombinant DNA methodologies, such that it is no longergenetically identical to the native cell or native tissue which isremoved from the organism. For example, a native autologous T-cell canbe genetically engineered by recombinant DNA techniques to become anautologous engineered cell expressing a transmembrane immunomodulatoryprotein and/or chimeric antigen receptor (CAR), which in some casesinvolves engineering a T-cell or TIL (tumor infiltrating lymphocyte).The engineered cells are then infused into a patient from which thenative T-cell was isolated. In some embodiments, the organism is humanor murine.

The terms “binding affinity,” and “binding avidity” as used herein meansthe specific binding affinity and specific binding avidity,respectively, of a protein for its counter-structure under specificbinding conditions. In biochemical kinetics avidity refers to theaccumulated strength of multiple affinities of individual non-covalentbinding interactions, such as between ICOSL and its counter-structuresICOS and/or CD28. As such, avidity is distinct from affinity, whichdescribes the strength of a single interaction. An increase orattenuation in binding affinity of a variant ICOSL containing anaffinity modified ICOSL IgSF domain to its counter-structure isdetermined relative to the binding affinity of the unmodified ICOSL,such as an unmodified ICOSL containing the native or wild-type IgSFdomain, such as IgV domain. Methods for determining binding affinity oravidity are known in art. See, for example, Larsen et al., AmericanJournal of Transplantation, Vol 5: 443-453 (2005). In some embodiments,a variant ICOSL of the invention (i.e. a ICOSL protein containing anaffinity modified IgSF domain) specifically binds to CD28 and/or ICOSmeasured by flow cytometry with a binding affinity that yields a MeanFluorescence Intensity (MFI) value at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 100% greater than a wild-type ICOSL control in abinding assay such as described in Example 6.

The term “biological half-life” refers to the amount of time it takesfor a substance, such as an immunomodulatory polypeptide comprising avariant ICOSL of the present invention, to lose half of itspharmacologic or physiologic activity or concentration. Biologicalhalf-life can be affected by elimination, excretion, degradation (e.g.,enzymatic) of the substance, or absorption and concentration in certainorgans or tissues of the body. In some embodiments, biological half-lifecan be assessed by determining the time it takes for the blood plasmaconcentration of the substance to reach half its steady state level(“plasma half-life”). Conjugates that can be used to derivatize andincrease the biological half-life of polypeptides of the invention areknown in the art and include, but are not limited to, polyethyleneglycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinantpeptides; see, WO2013130683), human serum albumin (HSA), bovine serumalbumin (BSA), lipids (acylation), and poly-Pro-Ala-Ser (PAS),polyglutamic acid (glutamylation).

The term “chimeric antigen receptor” or “CAR” as used herein refers toan artificial (i.e., man-made) transmembrane protein expressed on amammalian cell comprising at least an ectodomain, a transmembrane, andan endodomain. Optionally, the CAR protein includes a “spacer” whichcovalently links the ectodomain to the transmembrane domain. A spacer isoften a polypeptide linking the ectodomain to the transmembrane domainvia peptide bonds. The CAR is typically expressed on a mammalianlymphocyte. In some embodiments, the CAR is expressed on a mammaliancell such as a T-cell or a tumor infiltrating lymphocyte (TIL). A CARexpressed on a T-cell is referred to herein as a “CAR T-cell” or“CAR-T.” In some embodiments the CAR-T is a T helper cell, a cytotoxicT-cell, a natural killer T-cell, a memory T-cell, a regulatory T-cell,or a gamma delta T-cell. When used clinically in, e.g. adoptive celltransfer, a CAR-T with antigen binding specificity to the patient'stumor is typically engineered to express on a native T-cell obtainedfrom the patient. The engineered T-cell expressing the CAR is theninfused back into the patient. The CAR-T is thus often an autologousCAR-T although allogeneic CAR-T are included within the scope of theinvention. The ectodomain of a CAR comprises an antigen binding region,such as an antibody or antigen binding fragment thereof (e.g. scFv),that specifically binds under physiological conditions with a targetantigen, such as a tumor specific antigen. Upon specific binding abiochemical chain of events (i.e., signal transduction) results inmodulation of the immunological activity of the CAR-T. Thus, forexample, upon specific binding by the antigen binding region of theCAR-T to its target antigen can lead to changes in the immunologicalactivity of the T-cell activity as reflected by changes in cytotoxicity,proliferation or cytokine production. Signal transduction upon CAR-Tactivation is achieved in some embodiments by the CD3-zeta chain(“CD3-z”) which is involved in signal transduction in native mammalianT-cells. CAR-Ts can further comprises multiple signaling domains such asCD28, 41EE or OX40. to further modulate immunomodulatory response of theT-cell. CD3-z comprises a conserved motif known as an immunoreceptortyrosine-based activation motif (ITAM) which is involved in T-cellreceptor signal transduction.

The term “collectively” or “collective” when used in reference tocytokine production induced by the presence of two or more variant ICOSLof the invention in an in vitro assay, means the overall cytokineexpression level irrespective of the cytokine production induced byindividual variant ICOSL. In some embodiments, the cytokine beingassayed is IFN-gamma in an in vitro primary T-cell assay such asdescribed in Example 7.

The term “cognate binding partner” (used interchangeably with“counter-structure”) in reference to a polypeptide, such as in referenceto an IgSF domain of a variant ICOSL, refers to at least one molecule(typically a native mammalian protein) to which the referencedpolypeptide specifically binds under specific binding conditions. Insome aspects, a variant ICOSL containing an affinity modified IgSFdomain specifically binds to the counter-structure of the correspondingnative or wildtype ICOSL but with increased or attenuated affinity. Aspecies of ligand recognized and specifically binding to its cognatereceptor under specific binding conditions is an example of acounter-structure or cognate binding partner of that receptor. A“cognate cell surface binding partner” is a cognate binding partnerexpressed on a mammalian cell surface. A “cell surface molecularspecies” is a cognate binding partner of ligands of the immunologicalsynapse (IS), expressed on and by cells, such as mammalian cells,forming the immunological synapse.

As used herein, “conjugate,” “conjugation” or grammatical variationsthereof refers the joining or linking together of two or more compoundsresulting in the formation of another compound, by any joining orlinking methods known in the art. It can also refer to a compound whichis generated by the joining or linking together two or more compounds.For example, a variant ICOSL polypeptide linked directly or indirectlyto one or more chemical moieties or polypeptide is an exemplaryconjugate. Such conjugates include fusion proteins, those produced bychemical conjugates and those produced by any other methods.

The term “competitive binding” as used herein means that a protein iscapable of specifically binding to at least two cognate binding partnersbut that specific binding of one cognate binding partner inhibits, suchas prevents or precludes, simultaneous binding of the second cognatebinding partner. Thus, in some cases, it is not possible for a proteinto bind the two cognate binding partners at the same time. Generally,competitive binders contain the same or overlapping binding site forspecific binding but this is not a requirement. In some embodiments,competitive binding causes a measurable inhibition (partial or complete)of specific binding of a protein to one of its cognate binding partnerdue to specific binding of a second cognate binding partner. A varietyof methods are known to quantify competitive binding such as ELISA(enzyme linked immunosorbent assay) assays.

The term “conservative amino acid substitution” as used herein means anamino acid substitution in which an amino acid residue is substituted byanother amino acid residue having a side chain R group with similarchemical properties (e.g., charge or hydrophobicity). Examples of groupsof amino acids that have side chains with similar chemical propertiesinclude 1) aliphatic side chains: glycine, alanine, valine, leucine, andisoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3)amide-containing side chains: asparagine and glutamine; 4) aromatic sidechains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains:lysine, arginine, and histidine; 6) acidic side chains: aspartic acidand glutamic acid; and 7) sulfur-containing side chains: cysteine andmethionine. Conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.

The term, “corresponding to” with reference to positions of a protein,such as recitation that nucleotides or amino acid positions “correspondto” nucleotides or amino acid positions in a disclosed sequence, such asset forth in the Sequence listing, refers to nucleotides or amino acidpositions identified upon alignment with the disclosed sequence based onstructural sequence alignment or using a standard alignment algorithm,such as the GAP algorithm. For example, corresponding residues can bedetermined by alignment of a reference sequence with the sequence setforth in SEQ ID NO:32 (ECD domain) or set forth in SEQ ID NO: 196 (IgVdomain) by structural alignment methods as described herein. By aligningthe sequences, one skilled in the art can identify correspondingresidues, for example, using conserved and identical amino acid residuesas guides.

The terms “decrease” or “attenuate” “or suppress” as used herein meansto decrease by a statistically significant amount. A decrease can be atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

The terms “derivatives” or “derivatized” refer to modification of aprotein by covalently linking it, directly or indirectly, to acomposition so as to alter such characteristics as biological half-life,bioavailability, immunogenicity, solubility, toxicity, potency, orefficacy while retaining or enhancing its therapeutic benefit.Derivatives of immunomodulatory polypeptides of the invention are withinthe scope of the invention and can be made by, for example,glycosylation, pegylation, lipidation, or Fc-fusion.

As used herein, domain (typically a sequence of three or more, generally5 or 7 or more amino acids, such as 10 to 200 amino acid residues)refers to a portion of a molecule, such as a protein or encoding nucleicacid, that is structurally and/or functionally distinct from otherportions of the molecule and is identifiable. For example, domainsinclude those portions of a polypeptide chain that can form anindependently folded structure within a protein made up of one or morestructural motifs and/or that is recognized by virtue of a functionalactivity, such as binding activity. A protein can have one, or more thanone, distinct domains. For example, a domain can be identified, definedor distinguished by homology of the primary sequence or structure torelated family members, such as homology to motifs. In another example,a domain can be distinguished by its function, such as an ability tointeract with a biomolecule, such as a cognate binding partner. A domainindependently can exhibit a biological function or activity such thatthe domain independently or fused to another molecule can perform anactivity, such as, for example binding. A domain can be a linearsequence of amino acids or a non-linear sequence of amino acids. Manypolypeptides contain a plurality of domains. Such domains are known, andcan be identified by those of skill in the art. For exemplificationherein, definitions are provided, but it is understood that it is wellwithin the skill in the art to recognize particular domains by name. Ifneeded appropriate software can be employed to identify domains.

The term “ectodomain” as used herein refers to the region of a membraneprotein, such as a transmembrane protein, that lies outside thevesicular membrane. Ectodomains often comprise binding domains thatspecifically bind to ligands or cell surface receptors, such as via abinding domain that specifically binds to the ligand or cell surfacereceptor. The ectodomain of a cellular transmembrane protein isalternately referred to as an extracellular domain.

The terms “effective amount” or “therapeutically effective amount” referto a quantity and/or concentration of a therapeutic composition of theinvention, including a protein composition or cell composition, thatwhen administered ex vivo (by contact with a cell from a patient) or invivo (by administration into a patient) either alone (i.e., as amonotherapy) or in combination with additional therapeutic agents,yields a statistically significant decrease in disease progression as,for example, by ameliorating or eliminating symptoms and/or the cause ofthe disease. An effective amount may be an amount that relieves,lessens, or alleviates at least one symptom or biological response oreffect associated with a disease or disorder, prevents progression ofthe disease or disorder, or improves physical functioning of thepatient. In the case of cell therapy, the effective amount is aneffective dose or number of cells administered to a patient by adoptivecell therapy. In some embodiments the patient is a mammal such as anon-human primate or human patient.

The term “endodomain” as used herein refers to the region found in somemembrane proteins, such as transmembrane proteins, that extends into theinterior space defined by the cell surface membrane. In mammalian cells,the endodomain is the cytoplasmic region of the membrane protein. Incells, the endodomain interacts with intracellular constituents and canbe play a role in signal transduction and thus, in some cases, can be anintracellular signaling domain. The endodomain of a cellulartransmembrane protein is alternately referred to as a cytoplasmicdomain, which, in some cases, can be a cytoplasmic signaling domain.

The terms “enhanced” or “increased” as used herein in the context ofincreasing immunological activity of a mammalian lymphocyte means toincrease one or more activities the lymphocyte. An increased activitycan be one or more of increase cell survival, cell proliferation,cytokine production, or T-cell cytotoxicity, such as by a statisticallysignificant amount. In some embodiments, reference to increasedimmunological activity means to increase interferon gamma (IFN-gamma)production, such as by a statistically significant amount. In someembodiments, the immunological activity can be assessed in a mixedlymphocyte reaction (MLR) assay. Methods of conducting MLR assays areknown in the art. Wang et al., Cancer Immunol Res. 2014 September:2(9):846-56. Other methods of assessing activities of lymphocytes areknown in the art, including any assay as described herein. In someembodiments an enhancement can be an increase of at least 10%, 20%, 30%,40%, 50%, 75%,100%, 200%, 300%, 400%, or 500% greater than a non-zerocontrol value.

The term “engineered cell” as used herein refers to a mammalian cellthat has been genetically modified by human intervention such as byrecombinant DNA methods or viral transduction. In some embodiments, thecell is an immune cell, such as a lymphocyte (e.g. T cell, B cell, NKcell) or an antigen presenting cell (e.g. dendritic cell). The cell canbe a primary cell from a patient or can be a cell line. In someembodiments, an engineered cell of the invention comprises a variantICOSL of the invention engineered to modulate immunological activity ofa T-cell expressing CD28, ICOS, or CTLA-4 to which the variant ICOSLspecifically binds. In some embodiments, the variant ICOSL is atransmembrane immunomodulatory protein (hereinafter referred to as“TIP”) containing the extracellular domain or a portion thereofcontaining the IgV domain linked to a transmembrane domain (e.g. a ICOSLtransmembrane domain) and, optionally, an intracellular signalingdomain. In some cases, the TIP is formatted as a chimeric receptorcontaining a heterologous cytoplasmic signaling domain or endodomain. Insome embodiments, an engineered cell is capable of expressing andsecreting a immunomodulatory protein as described herein. Among providedengineered cells also are cells further containing an engineered T-cellreceptor (TCR) or chimeric antigen receptor (CAR).

The term “engineered T-cell” as used herein refers to a T-cell such as aT helper cell, cytotoxic T-cell (alternatively, cytotoxic T lymphocyteor CTL), natural killer T-cell, regulatory T-cell, memory T-cell, orgamma delta T-cell, that has been genetically modified by humanintervention such as by recombinant DNA methods or viral transductionmethods. An engineered T-cell comprises a variant ICOSL transmembraneimmunomodulatory protein (TIP) of the present invention that isexpressed on the T-cell and is engineered to modulate immunologicalactivity of the engineered T-cell itself, or a mammalian cell to whichthe variant ICOSL expressed on the T-cell specifically binds. The term“engineered T-cell receptor” or “engineered TCR” refers to a T-cellreceptor (TCR) engineered to specifically bind with a desired affinityto a major histocompatibility complex (MHC)/peptide target antigen thatis selected, cloned, and/or subsequently introduced into a population ofT-cells, often used for adoptive immunotherapy. In contrast toengineered TCRs, CARs are engineered to bind target antigens in a MHCindependent manner.

The term “expressed on” as used herein is used in reference to a proteinexpressed on the surface of a cell, such as a mammalian cell. Thus, theprotein is expressed as a membrane protein. In some embodiments, theexpressed protein is a transmembrane protein. In some embodiments, theprotein is conjugated to a small molecule moiety such as a drug ordetectable label. Proteins expressed on the surface of a cell caninclude cell-surface proteins such as cell surface receptors that areexpressed on mammalian cells.

The term “half-life extending moiety” refers to a moiety of apolypeptide fusion or chemical conjugate that extends the half-life of aprotein circulating in mammalian blood serum compared to the half-lifeof the protein that is not so conjugated to the moiety. In someembodiments, half-life is extended by greater than or greater than about1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold., 5.0-fold, or6.0-fold. In some embodiments, half-life is extended by more than 6hours, more than 12 hours, more than 24 hours, more than 48 hours, morethan 72 hours, more than 96 hours or more than 1 week after in vivoadministration compared to the protein without the half-life extendingmoiety. The half-life refers to the amount of time it takes for theprotein to lose half of its concentration, amount, or activity.Half-life can be determined for example, by using an ELISA assay or anactivity assay. Exemplary half-life extending moieties include an Fcdomain, a multimerization domain, polyethylene glycol (PEG),hydroxyethyl starch (HES), XTEN (extended recombinant peptides; see,WO2013130683), human serum albumin (HSA), bovine serum albumin (BSA),lipids (acylation), and poly-Pro-Ala-Ser (PAS), and polyglutamic acid(glutamylation).

The term “immunological synapse” or “immune synapse” as used hereinmeans the interface between a mammalian cell that expresses MHC I (majorhistocompatibility complex) or MHC II, such as an antigen-presentingcell or tumor cell, and a mammalian lymphocyte such as an effector Tcell or Natural Killer (NK) cell.

An Fc (fragment crystallizable) region or domain of an immunoglobulinmolecule (also termed an Fc polypeptide) corresponds largely to theconstant region of the immunoglobulin heavy chain, and is responsiblefor various functions, including the antibody's effector function(s).The Fc domain contains part or all of a hinge domain of animmunoglobulin molecule plus a CH2 and a CH3 domain. The Fc domain canform a dimer of two polypeptide chains joined by one or more disulfidebonds. In some embodiments, the Fc is a variant Fc that exhibits reduced(e.g. reduced greater than 30%, 40%, 50%, 60%, 70%, 80%, 90% or more)activity to facilitate an effector function. In some embodiments,reference to amino acid substitutions in an Fc region is by EU numberingsystem unless described with reference to a specific SEQ ID NO. EUnumbering is known and is according to the most recently updated IMGTScientific Chart (IMGT®, the international ImMunoGeneTics informationsystem®http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html(created: 17 May 2001, last updated: 10 Jan. 2013) and the EU index asreported in Kabat, E. A. et al. Sequences of Proteins of Immunologicalinterest. 5th ed. US Department of Health and Human Services, NIHpublication No. 91-3242 (1991).

An immunoglobulin Fc fusion (“Fc-fusion”), such as an immunomodulatoryFc fusion protein, is a molecule comprising one or more polypeptides (orone or more small molecules) operably linked to an Fc region of animmunoglobulin. An Fc-fusion may comprise, for example, the Fc region ofan antibody (which facilitates effector functions and pharmacokinetics)and a variant ICOSL. An immunoglobulin Fc region may be linkedindirectly or directly to one or more variant ICOSL or small molecules(fusion partners). Various linkers are known in the art and canoptionally be used to link an Fc to a fusion partner to generate anFc-fusion. Fc-fusions of identical species can be dimerized to formFc-fusion homodimers, or using non-identical species to form Fc-fusionheterodimers. In some embodiments, the Fc is a mammalian Fc such as amurine or human Fc.

The term “host cell” refers to a cell that can be used to express aprotein encoded by a recombinant expression vector. A host cell can be aprokaryote, for example, E. coli, or it can be a eukaryote, for example,a single-celled eukaryote (e.g., a yeast or other fungus), a plant cell(e.g., a tobacco or tomato plant cell), an animal cell (e.g., a humancell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or aninsect cell) or a hybridoma. Examples of host cells include Chinesehamster ovary (CHO) cells or their derivatives such as Veggie CHO andrelated cell lines which grow in serum-free media or CHO strain DX-B 11,which is deficient in DHFR. In some embodiments, a host cell is amammalian cell (e.g., a human cell, a monkey cell, a hamster cell, a ratcell, a mouse cell, or an insect cell).

The term “immunoglobulin” (abbreviated “Ig”) as used herein refers to amammalian immunoglobulin protein including any of the five human classesof antibody: IgA (which includes subclasses IgA1 and IgA2), IgD, IgE,IgG (which includes subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. Theterm is also inclusive of immunoglobulins that are less thanfull-length, whether wholly or partially synthetic (e.g., recombinant orchemical synthesis) or naturally produced, such as antigen bindingfragment (Fab), variable fragment (Fv) containing V_(H) and V_(L), thesingle chain variable fragment (scFv) containing V_(H) and V_(L) linkedtogether in one chain, as well as other antibody V region fragments,such as Fab′, F(ab)₂, F(ab′)₂, dsFv diabody, Fc, and Fd polypeptidefragments. Bispecific antibodies, homobispecific and heterobispecific,are included within the meaning of the term.

The term “immunoglobulin superfamily” or “IgSF” as used herein means thegroup of cell surface and soluble proteins that are involved in therecognition, binding, or adhesion processes of cells. Molecules arecategorized as members of this superfamily based on shared structuralfeatures with immunoglobulins (i.e., antibodies); they all possess adomain known as an immunoglobulin domain or fold. Members of the IgSFinclude cell surface antigen receptors, co-receptors and co-stimulatorymolecules of the immune system, molecules involved in antigenpresentation to lymphocytes, cell adhesion molecules, certain cytokinereceptors and intracellular muscle proteins. They are commonlyassociated with roles in the immune system. Proteins in theimmunological synapse are often members of the IgSF. IgSF can also beclassified into “subfamilies” based on shared properties such asfunction. Such subfamilies typically consist of from 4 to 30 IgSFmembers.

The terms “IgSF domain” or “immunoglobulin domain” or “Ig domain” asused herein refers to a structural domain of IgSF proteins. Ig domainsare named after the immunoglobulin molecules. They contain about 70-110amino acids and are categorized according to their size and function.Ig-domains possess a characteristic Ig-fold, which has a sandwich-likestructure formed by two sheets of antiparallel beta strands.Interactions between hydrophobic amino acids on the inner side of thesandwich and highly conserved disulfide bonds formed between cysteineresidues in the B and F strands, stabilize the Ig-fold. One end of theIg domain has a section called the complementarity determining regionthat is important for the specificity of antibodies for their ligands.The Ig like domains can be classified (into classes) as: IgV, IgC1,IgC2, or IgI. Most Ig domains are either variable (IgV) or constant(IgC). IgV domains with 9 beta strands are generally longer than IgCdomains with 7 beta strands. Ig domains of some members of the IgSFresemble IgV domains in the amino acid sequence, yet are similar in sizeto IgC domains. These are called IgC2 domains, while standard IgCdomains are called IgC1 domains. T-cell receptor (TCR) chains containtwo Ig domains in the extracellular portion; one IgV domain at theN-terminus and one IgC1 domain adjacent to the cell membrane. ICOSLcontains two Ig domains: IgV and IgC.

The term “IgSF species” as used herein means an ensemble of IgSF memberproteins with identical or substantially identical primary amino acidsequence. Each mammalian immunoglobulin superfamily (IgSF) memberdefines a unique identity of all IgSF species that belong to that IgSFmember. Thus, each IgSF family member is unique from other IgSF familymembers and, accordingly, each species of a particular IgSF familymember is unique from the species of another IgSF family member.Nevertheless, variation between molecules that are of the same IgSFspecies may occur owing to differences in post-translationalmodification such as glycosylation, phosphorylation, ubiquitination,nitrosylation, methylation, acetylation, and lipidation. Additionally,minor sequence differences within a single IgSF species owing to genepolymorphisms constitute another form of variation within a single IgSFspecies as do wild type truncated forms of IgSF species owing to, forexample, proteolytic cleavage. A “cell surface IgSF species” is an IgSFspecies expressed on the surface of a cell, generally a mammalian cell.

The term “immunological activity” as used herein in the context ofmammalian lymphocytes such as T-cells refers to one or more cellsurvival, cell proliferation, cytokine production (e.g.interferon-gamma), or T-cell cytotoxicity activities. In some cases, animmunological activity can mean the cell expression of cytokines, suchas chemokines or interleukins. Assays for determining enhancement orsuppression of immunological activity include the MLR (mixed lymphocytereaction) assays measuring interferon-gamma cytokine levels in culturesupernatants (Wang et al., Cancer Immunol Res. 2014 September:2(9):846-56), SEB (staphylococcal enterotoxin B) T cell stimulationassay (Wang et al., Cancer Immunol Res. 2014 September: 2(9):846-56),and anti-CD3 T cell stimulation assays (Li and Kurlander, J Transl Med.2010: 8: 104). Since T cell activation is associated with secretion ofIFN-gamma cytokine, detecting IFN-gamma levels in culture supernatantsfrom these in vitro human T cell assays can be assayed using commercialELISA kits (Wu et al, Immunol Lett 2008 Apr. 15; 117(1): 57-62).Induction of an immune response results in an increase in immunologicalactivity relative to quiescent lymphocytes. An immunomodulatory protein,such as a variant ICOSL polypeptide containing an affinity modified IgSFdomain, as provided herein can in some embodiments increase or, inalternative embodiments, decrease IFN-gamma (interferon-gamma)expression in a primary T-cell assay relative to a wild-type IgSF memberor IgSF domain control. Those of skill will recognize that the format ofthe primary T-cell assay used to determine an increase in IFN-gammaexpression will differ from that employed to assay for a decrease inIFN-gamma expression. In assaying for the ability of an immunomodulatoryprotein or affinity modified IgSF domain of the invention to decreaseIFN-gamma expression in a primary T-cell assay, a Mixed LymphocyteReaction (MLR) assay can be used as described in Example 6.Conveniently, a soluble form of an affinity modified IgSF domain of theinvention can be employed to determine its ability to antagonize andthereby decrease the IFN-gamma expression in a MLR as likewise describedin Example 6. Alternatively, in assaying for the ability of animmunomodulatory protein or affinity modified IgSF domain of theinvention to increase IFN-gamma expression in a primary T-cell assay, aco-immobilization assay can be used. In a co-immobilization assay, aT-cell receptor signal, provided in some embodiments by anti-CD3antibody, is used in conjunction with a co-immobilized affinity modifiedIgSF domain, such as variant ICOSL, to determine the ability to increaseIFN-gamma expression relative to a wild-type IgSF domain control.Methods to assay the immunological activity of engineered cells,including to evaluate the activity of a variant ICOSL transmembraneimmunomodulatory protein, are known in the art and include, but are notlimited to, the ability to expand T cells following antigen stimulation,sustain T cell expansion in the absence of re- stimulation, andanti-cancer activities in appropriate animal models. Assays also includeassays to assess cytotoxicity, including a standard ⁵¹Cr-release assay(see e.g. Milone et al., (2009) Molecular Therapy 17: 1453-1464) or flowbased cytotoxicity assays, or an impedance based cytotoxicity assay(Peper et al. (2014) Journal of Immunological Methods, 405:192-198).

An “immunomodulatory polypeptide” is a polypeptide that modulatesimmunological activity. By “modulation” or “modulating” an immuneresponse is meant that immunological activity is either increased ordecreased. An immunomodulatory polypeptide can be a single polypeptidechain or a multimer (dimers or higher order multimers) of at least twopolypeptide chains covalently bonded to each other by, for example,interchain disulfide bonds. Thus, monomeric, dimeric, and higher ordermultimeric polypeptides are within the scope of the defined term.Multimeric polypeptides can be homomultimeric (of identical polypeptidechains) or heteromultimeric (of non-identical polypeptide chains). Animmunomodulatory polypeptide of the invention comprises a variant ICOSL.

The term “increase” as used herein means to increase by a statisticallysignificant amount. An increase can be at least 5%, 10%, 20%, 30%, 40%,50%, 75%, 100%, or greater than a non-zero control value.

An “isoform” of ICOSL (inducible costimulator ligand; CD275) is one of aplurality naturally occurring ICOSL polypeptides that differ in aminoacid sequence. Isoforms can be the product of splice variants of an RNAtranscript expressed by a single gene, or the expression product ofhighly similar but different genes yielding a functionally similarprotein such as may occur from gene duplication. As used herein, theterm “isoform” of ICOSL also refers to the product of different allelesof an ICOSL gene (e.g., ICOSLG).

The term “lymphocyte” as used herein means any of three subtypes ofwhite blood cell in a mammalian immune system. They include naturalkiller cells (NK cells) (which function in cell-mediated, cytotoxicinnate immunity), T cells (for cell-mediated, cytotoxic adaptiveimmunity), and B cells (for humoral, antibody-driven adaptive immunity).T cells include: T helper cells, cytotoxic T-cells, natural killerT-cells, memory T-cells, regulatory T-cells, or gamma delta T-cells.Innate lymphoid cells (ILC) are also included within the definition oflymphocyte.

The terms “mammal,” or “patient” specifically includes reference to atleast one of a: human, chimpanzee, rhesus monkey, cynomolgus monkey,dog, cat, mouse, or rat.

The term “membrane protein” as used herein means a protein that, underphysiological conditions, is attached directly or indirectly to a lipidbilayer. A lipid bilayer that forms a membrane can be a biologicalmembrane such as a eukaryotic (e.g., mammalian) cell membrane or anartificial (i.e., man-made) membrane such as that found on a liposome.Attachment of a membrane protein to the lipid bilayer can be by way ofcovalent attachment, or by way of non-covalent interactions such ashydrophobic or electrostatic interactions. A membrane protein can be anintegral membrane protein or a peripheral membrane protein. Membraneproteins that are peripheral membrane proteins are non-covalentlyattached to the lipid bilayer or non-covalently attached to an integralmembrane protein. A peripheral membrane protein forms a temporaryattachment to the lipid bilayer such that under the range of conditionsthat are physiological in a mammal, peripheral membrane protein canassociate and/or disassociate from the lipid bilayer. In contrast toperipheral membrane proteins, integral membrane proteins form asubstantially permanent attachment to the membrane's lipid bilayer suchthat under the range of conditions that are physiological in a mammal,integral membrane proteins do not disassociate from their attachment tothe lipid bilayer. A membrane protein can form an attachment to themembrane by way of one layer of the lipid bilayer (monotopic), orattached by way of both layers of the membrane (polytopic). An integralmembrane protein that interacts with only one lipid bilayer is an“integral monotopic protein”. An integral membrane protein thatinteracts with both lipid bilayers is an “integral polytopic protein”alternatively referred to herein as a “transmembrane protein”.

The terms “modulating” or “modulate” as used herein in the context of animmune response, such as a mammalian immune response, refer to anyalteration, such as an increase or a decrease, of existing or potentialimmune responses that occurs as a result of administration of animmunomodulatory polypeptide comprising a variant ICOSL of the presentinvention or as a result of administration of engineered cells expressesan immunomodulatory protein, such as a variant ICOSL transmembraneimmunomodulatory protein of the present invention. Thus, it refers to analteration, such as an increase or decrease, of an immune response ascompared to the immune response that occurs or is present in the absenceof the administration of the immunomodulatory protein comprising thevariant ICOSL or cells expressing such an immunomodulatory polypeptide.Such modulation includes any induction, activation, suppression oralteration in degree or extent of immunological activity of an immunecell. Immune cells include B cells, T cells, NK (natural killer) cells,NK T cells, professional antigen-presenting cells (APCs), andnon-professional antigen-presenting cells, and inflammatory cells(neutrophils, macrophages, monocytes, eosinophils, and basophils).Modulation includes any change imparted on an existing immune response,a developing immune response, a potential immune response, or thecapacity to induce, regulate, influence, or respond to an immuneresponse. Modulation includes any alteration in the expression and/orfunction of genes, proteins and/or other molecules in immune cells aspart of an immune response. Modulation of an immune response ormodulation of immunological activity includes, for example, thefollowing: elimination, deletion, or sequestration of immune cells;induction or generation of immune cells that can modulate the functionalcapacity of other cells such as autoreactive lymphocytes, antigenpresenting cells, or inflammatory cells; induction of an unresponsivestate in immune cells (i.e., anergy); enhancing or suppressing theactivity or function of immune cells, including but not limited toaltering the pattern of proteins expressed by these cells. Examplesinclude altered production and/or secretion of certain classes ofmolecules such as cytokines, chemokines, growth factors, transcriptionfactors, kinases, costimulatory molecules, or other cell surfacereceptors or any combination of these modulatory events. Modulation canbe assessed, for example, by an alteration in IFN-gamma (interferongamma) expression relative to the wild-type ICOSL control in a primary Tcell assay (see, Zhao and Ji, Exp Cell Res. 2016 Jan. 1; 340(1)132-138). Modulation can be assessed, for example, by an alteration ofan immunological activity of engineered cells, such as an alteration inin cytotoxic activity of engineered cells or an alteration in cytokinesecretion of engineered cells relative to cells engineered with awild-type ICOSL transmembrane protein.

The term “molecular species” as used herein means an ensemble ofproteins with identical or substantially identical primary amino acidsequence. Each mammalian immunoglobulin superfamily (IgSF) memberdefines a collection of identical or substantially identical molecularspecies. Thus, for example, human ICOSL is an IgSF member and each humanICOSL molecule is a molecule species of ICOS. Variation betweenmolecules that are of the same molecular species may occur owing todifferences in post-translational modification such as glycosylation,phosphorylation, ubiquitination, nitrosylation, methylation,acetylation, and lipidation. Additionally, minor sequence differenceswithin a single molecular species owing to gene polymorphisms constituteanother form of variation within a single molecular species as do wildtype truncated forms of a single molecular species owing to, forexample, proteolytic cleavage. A “cell surface molecular species” is amolecular species expressed on the surface of a mammalian cell. Two ormore different species of protein, each of which is present exclusivelyon one or exclusively the other (but not both) of the two mammaliancells forming the IS, are said to be in “cis” or “cis configuration”with each other. Two different species of protein, the first of which isexclusively present on one of the two mammalian cells forming the IS andthe second of which is present exclusively on the second of the twomammalian cells forming the IS, are said to be in “trans” or “transconfiguration.” Two different species of protein each of which ispresent on both of the two mammalian cells forming the IS are in bothcis and trans configurations on these cells.

The term, a “multimerization domain” refers to a sequence of amino acidsthat promotes stable interaction of a polypeptide molecule with one ormore additional polypeptide molecules, each containing a complementarymultimerization domain, which can be the same or a differentmultimerization domain to form a stable multimer with the first domain.Generally, a polypeptide is joined directly or indirectly to themultimerization domain. Exemplary multimerization domains include theimmunoglobulin sequences or portions thereof, leucine zippers,hydrophobic regions, hydrophilic regions, and compatible protein-proteininteraction domains. The multimerization domain, for example, can be animmunoglobulin constant region or domain, such as, for example, the Fcdomain or portions thereof from IgG, including IgG1, IgG2, IgG3 or IgG4subtypes, IgA, IgE, IgD and IgM and modified forms thereof.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyto refer to a polymer of nucleic acid residues (e.g.,deoxyribonucleotides or ribonucleotides) in either single- ordouble-stranded form. Unless specifically limited, the terms encompassnucleic acids containing known analogues of natural nucleotides and thathave similar binding properties to it and are metabolized in a mannersimilar to naturally-occurring nucleotides. Unless otherwise indicated,a particular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary nucleotide sequences as well as thesequence explicitly indicated (a “reference sequence”). Specifically,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues. The termnucleic acid or polynucleotide encompasses cDNA or mRNA encoded by agene.

The term “non-competitive binding” as used herein means the ability of aprotein to specifically bind simultaneously to at least two cognatebinding partners. Thus, the protein is able to bind to at least twodifferent cognate binding partners at the same time, although thebinding interaction need not be for the same duration such that, in somecases, the protein is specifically bound to only one of the cognatebinding partners. In some embodiments, the binding occurs under specificbinding conditions. In some embodiments, the simultaneous binding issuch that binding of one cognate binding partner does not substantiallyinhibit simultaneous binding to a second cognate binding partner. Insome embodiments, non-competitive binding means that binding a secondcognate binding partner to its binding site on the protein does notdisplace the binding of a first cognate binding partner to its bindingsite on the protein. Methods of assessing non-competitive binding arewell known in the art such as the method described in Perez de La Lastraet al., Immunology, 1999 April: 96(4): 663-670. In some cases, innon-competitive interactions, the first cognate binding partnerspecifically binds at an interaction site that does not overlap with theinteraction site of the second cognate binding partner such that bindingof the second cognate binding partner does not directly interfere withthe binding of the first cognate binding partner. Thus, any effect onbinding of the cognate binding partner by the binding of the secondcognate binding partner is through a mechanism other than directinterference with the binding of the first cognate binding partner. Forexample, in the context of enzyme-substrate interactions, anon-competitive inhibitor binds to a site other than the active site ofthe enzyme. Non-competitive binding encompasses uncompetitive bindinginteractions in which a second cognate binding partner specificallybinds at an interaction site that does not overlap with the binding ofthe first cognate binding partner but binds to the second interactionsite only when the first interaction site is occupied by the firstcognate binding partner.

The term “pharmaceutical composition” refers to a composition suitablefor pharmaceutical use in a mammalian subject, often a human. Apharmaceutical composition typically comprises an effective amount of anactive agent (e.g., an immunomodulatory polypeptide comprising a variantICOSL or engineered cells expressing a variant ICOSL transmembraneimmunomodulatory protein) and a carrier, excipient, or diluent. Thecarrier, excipient, or diluent is typically a pharmaceuticallyacceptable carrier, excipient or diluent, respectively.

The terms “polypeptide” and “protein” are used interchangeably hereinand refer to a molecular chain of two or more amino acids linked throughpeptide bonds. The terms do not refer to a specific length of theproduct. Thus, “peptides,” and “oligopeptides,” are included within thedefinition of polypeptide. The terms include post-translationalmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like. The terms also includemolecules in which one or more amino acid analogs or non-canonical orunnatural amino acids are included as can be synthesized, or expressedrecombinantly using known protein engineering techniques. In addition,proteins can be derivatized.

The term “primary T-cell assay” as used herein refers to an in vitroassay to measure interferon-gamma (“IFN-gamma”) expression. A variety ofsuch primary T-cell assays are known in the art such as that describedin Example 7. In a preferred embodiment, the assay used is an anti-CD3coimmobilizaton assay. In this assay, primary T cells are stimulated byanti-CD3 immobilized with or without additional recombinant proteins.Culture supernatants are harvested at timepoints, usually 24-72 hours.In another embodiment, the assay used is the MLR. In this assay, primaryT cells are stimulated with allogeneic APC. Culture supernatants areharvested at timepoints, usually 24-72 hours Human IFN-gamma levels aremeasured in culture supernatants by standard ELISA techniques.Commercial kits are available from vendors and the assay is performedaccording to manufacturer's recommendation.

The term “purified” as applied to nucleic acids, such as encodingimmunomodulatory proteins of the invention, generally denotes a nucleicacid or polypeptide that is substantially free from other components asdetermined by analytical techniques well known in the art (e.g., apurified polypeptide or polynucleotide forms a discrete band in anelectrophoretic gel, chromatographic eluate, and/or a media subjected todensity gradient centrifugation). For example, a nucleic acid orpolypeptide that gives rise to essentially one band in anelectrophoretic gel is “purified.” A purified nucleic acid or protein ofthe invention is at least about 50% pure, usually at least about 75%,80%, 85%, 90%, 95%, 96%, 99% or more pure (e.g., percent by weight or ona molar basis).

The term “recombinant” indicates that the material (e.g., a nucleic acidor a polypeptide) has been artificially (i.e., non-naturally) altered byhuman intervention. The alteration can be performed on the materialwithin, or removed from, its natural environment or state. For example,a “recombinant nucleic acid” is one that is made by recombining nucleicacids, e.g., during cloning, affinity modification, DNA shuffling orother well-known molecular biological procedures. A “recombinant DNAmolecule,” is comprised of segments of DNA joined together by means ofsuch molecular biological techniques. The term “recombinant protein” or“recombinant polypeptide” as used herein refers to a protein moleculewhich is expressed using a recombinant DNA molecule. A “recombinant hostcell” is a cell that contains and/or expresses a recombinant nucleicacid or that is otherwise altered by genetic engineering, such as byintroducing into the cell a nucleic acid molecule encoding a recombinantprotein, such as a transmembrane immunomodulatory protein providedherein. Transcriptional control signals in eukaryotes comprise“promoter” and “enhancer” elements. Promoters and enhancers consist ofshort arrays of DNA sequences that interact specifically with cellularproteins involved in transcription. Promoter and enhancer elements havebeen isolated from a variety of eukaryotic sources including genes inyeast, insect and mammalian cells and viruses (analogous controlelements, i.e., promoters, are also found in prokaryotes). The selectionof a particular promoter and enhancer depends on what cell type is to beused to express the protein of interest. The terms “in operablecombination,” “in operable order” and “operably linked” as used hereinrefer to the linkage of nucleic acid sequences in such a manner ororientation that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced.

The term “recombinant expression vector” as used herein refers to a DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host cell. Nucleic acid sequencesnecessary for expression in prokaryotes include a promoter, optionallyan operator sequence, a ribosome binding site and possibly othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals. A secretory signal peptidesequence can also, optionally, be encoded by the recombinant expressionvector, operably linked to the coding sequence for the recombinantprotein, such as a recombinant fusion protein, so that the expressedfusion protein can be secreted by the recombinant host cell, for easierisolation of the fusion protein from the cell, if desired. The termincludes the vector as a self-replicating nucleic acid structure as wellas the vector incorporated into the genome of a host cell into which ithas been introduced. Among the vectors are viral vectors, such aslentiviral vectors.

The term “selectivity” refers to the preference of a subject protein, orpolypeptide, for specific binding of one substrate, such as one cognatebinding partner, compared to specific binding for another substrate,such as a different cognate binding partner of the subject protein.Selectivity can be reflected as a ratio of the binding activity (e.g.binding affinity) of a subject protein and a first substrate, such as afirst cognate binding partner, (e.g., K_(d1)) and the binding activity(e.g. binding affinity) of the same subject protein with a secondcognate binding partner (e.g., K_(d2)).

The term “sequence identity” as used herein refers to the sequenceidentity between genes or proteins at the nucleotide or amino acidlevel, respectively. “Sequence identity” is a measure of identitybetween proteins at the amino acid level and a measure of identitybetween nucleic acids at nucleotide level. The protein sequence identitymay be determined by comparing the amino acid sequence in a givenposition in each sequence when the sequences are aligned. Similarly, thenucleic acid sequence identity may be determined by comparing thenucleotide sequence in a given position in each sequence when thesequences are aligned. Methods for the alignment of sequences forcomparison are well known in the art, such methods include GAP, BESTFIT,BLAST, FASTA and TFASTA. The BLAST algorithm calculates percent sequenceidentity and performs a statistical analysis of the similarity betweenthe two sequences. The software for performing BLAST analysis ispublicly available through the National Center for BiotechnologyInformation (NCBI) website.

The term “soluble” as used herein in reference to proteins, means thatthe protein is not a membrane protein. In general, a soluble proteincontains only the extracellular domain of an IgSF family memberreceptor, or a portion thereof containing an IgSF domain or domains orspecific-binding fragments thereof, but does not contain thetransmembrane domain. In some cases, solubility of a protein can beimproved by linkage or attachment, directly or indirectly via a linker,to an Fc domain, which, in some cases, also can improve the stabilityand/or half-life of the protein. In some aspects, a soluble protein isan Fc fusion protein.

The term “species” as used herein with respect to polypeptides ornucleic acids means an ensemble of molecules with identical orsubstantially identical sequences. Variation between polypeptides thatare of the same species may occur owing to differences inpost-translational modification such as glycosylation, phosphorylation,ubiquitination, nitrosylation, methylation, acetylation, and lipidation.Slightly truncated sequences of polypeptides that differ (or encode adifference) from the full length species at the amino-terminus orcarboxy-terminus by no more than 1, 2, or 3 amino acid residues areconsidered to be of a single species. Such microheterogeneities are acommon feature of manufactured proteins.

The term “specific binding fragment” as used herein in reference to afull-length wild-type mammalian ICOSL polypeptide or an IgV or an IgCdomain thereof, means a polypeptide having a subsequence of an IgVand/or IgC domain and that specifically binds in vitro and/or in vivo toa mammalian ICOS and/or mammalian CD28 such as a human or murine ICOS orCD28. In some embodiments, the specific binding fragment of ICOSL IgV orICOSL IgC is at at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% the sequence length of the full-length wild-type sequence.The specific binding fragment can be altered in sequence to form avariant ICOSL of the invention.

The term “specifically binds” as used herein means the ability of aprotein, under specific binding conditions, to bind to a target proteinsuch that its affinity or avidity is at least 5 times as great, butoptionally at least 10, 20, 30, 40, 50, 100, 250 or 500 times as great,or even at least 1000 times as great as the average affinity or avidityof the same protein to a collection of random peptides or polypeptidesof sufficient statistical size. A specifically binding protein need notbind exclusively to a single target molecule but may specifically bindto a non-target molecule due to similarity in structural conformationbetween the target and non-target (e.g., paralogs or orthologs). Thoseof skill will recognize that specific binding to a molecule having thesame function in a different species of animal (i.e., ortholog) or to anon-target molecule having a substantially similar epitope as the targetmolecule (e.g., paralog) is possible and does not detract from thespecificity of binding which is determined relative to a statisticallyvalid collection of unique non-targets (e.g., random polypeptides).Thus, a polypeptide of the invention may specifically bind to more thanone distinct species of target molecule due to cross-reactivity.Solid-phase ELISA immunoassays or Biacore measurements can be used todetermine specific binding between two proteins. Generally, interactionsbetween two binding proteins have dissociation constants (K_(d)) lessthan 1×10⁻⁵ M, and often as low as 1×10⁻¹² M. In certain embodiments ofthe present disclosure, interactions between two binding proteins havedissociation constants of 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M,1×10⁻¹⁰ M or 1×10⁻¹¹ M.

The terms “surface expresses” or “surface expression” in reference to amammalian cell expressing a polypeptide means that the polypeptide isexpressed as a membrane protein. In some embodiments, the membraneprotein is a transmembrane protein.

As used herein, “synthetic,” with reference to, for example, a syntheticnucleic acid molecule or a synthetic gene or a synthetic peptide refersto a nucleic acid molecule or polypeptide molecule that is produced byrecombinant methods and/or by chemical synthesis methods.

The term “targeting moiety” as used herein refers to a composition thatis covalently or non-covalently attached to, or physically encapsulates,a polypeptide comprising a variant ICOSL of the present invention. Thetargeting moiety has specific binding affinity for a desiredcounter-structure such as a cell surface receptor (e.g., the B7 familymember PD-L1), or a tumor antigen such as tumor specific antigen (TSA)or a tumor associated antigen (TAA) such as B7-H6. Typically, thedesired counter-structure is localized on a specific tissue orcell-type. Targeting moieties include: antibodies, antigen bindingfragment (Fab), variable fragment (Fv) containing V_(H) and V_(L), thesingle chain variable fragment (scFv) containing V_(H) and V_(L) linkedtogether in one chain, as well as other antibody V region fragments,such as Fab′, F(ab)₂, F(ab′)₂, dsFv diabody, nanobodies, solublereceptors, receptor ligands, affinity matured receptors or ligands, aswell as small molecule (<500 dalton) compositions (e.g., specificbinding receptor compositions). Targeting moieties can also be attachedcovalently or non-covalently to the lipid membrane of liposomes thatencapsulate a polypeptide of the present invention.

The term “transmembrane protein” as used herein means a membrane proteinthat substantially or completely spans a lipid bilayer such as thoselipid bilayers found in a biological membrane such as a mammalian cell,or in an artificial construct such as a liposome. The transmembraneprotein comprises a transmembrane domain (“transmembrane domain”) bywhich it is integrated into the lipid bilayer and by which theintegration is thermodynamically stable under physiological conditions.Transmembrane domains are generally predictable from their amino acidsequence via any number of commercially available bioinformaticssoftware applications on the basis of their elevated hydrophobicityrelative to regions of the protein that interact with aqueousenvironments (e.g., cytosol, extracellular fluid). A transmembranedomain is often a hydrophobic alpha helix that spans the membrane. Atransmembrane protein can pass through the both layers of the lipidbilayer once or multiple times. A transmembrane protein includes theprovided transmembrane immunomodulatory proteins described herein. Inaddition to the transmembrane domain, a transmembrane immunomodulatoryprotein of the invention further comprises an ectodomain and, in someembodiments, an endodomain.

The terms “treating,” “treatment,” or “therapy” of a disease or disorderas used herein mean slowing, stopping or reversing the disease ordisorders progression, as evidenced by decreasing, cessation orelimination of either clinical or diagnostic symptoms, by administrationof a therapeutic composition (e.g. containing an immunomodulatoryprotein or engineered cells) of the invention either alone or incombination with another compound as described herein. “Treating,”“treatment,” or “therapy” also means a decrease in the severity ofsymptoms in an acute or chronic disease or disorder or a decrease in therelapse rate as for example in the case of a relapsing or remittingautoimmune disease course or a decrease in inflammation in the case ofan inflammatory aspect of an autoimmune disease. As used herein in thecontext of cancer, the terms “treatment” or, “inhibit,” “inhibiting” or“inhibition” of cancer refers to at least one of: a statisticallysignificant decrease in the rate of tumor growth, a cessation of tumorgrowth, or a reduction in the size, mass, metabolic activity, or volumeof the tumor, as measured by standard criteria such as, but not limitedto, the Response Evaluation Criteria for Solid Tumors (RECIST), or astatistically significant increase in progression free survival (PFS) oroverall survival (OS). “Preventing,” “prophylaxis,” or “prevention” of adisease or disorder as used in the context of this invention refers tothe administration of an immunomodulatory polypeptide or engineeredcells of the invention, either alone or in combination with anothercompound, to prevent the occurrence or onset of a disease or disorder orsome or all of the symptoms of a disease or disorder or to lessen thelikelihood of the onset of a disease or disorder.

The term “tumor specific antigen” or “TSA” as used herein refers to acounter-structure that is present primarily on tumor cells of amammalian subject but generally not found on normal cells of themammalian subject. A tumor specific antigen need not be exclusive totumor cells but the percentage of cells of a particular mammal that havethe tumor specific antigen is sufficiently high or the levels of thetumor specific antigen on the surface of the tumor are sufficiently highsuch that it can be targeted by anti-tumor therapeutics, such asimmunomodulatory polypeptides of the invention, and provide preventionor treatment of the mammal from the effects of the tumor. In someembodiments, in a random statistical sample of cells from a mammal witha tumor, at least 50% of the cells displaying a TSA are cancerous. Inother embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of thecells displaying a TSA are cancerous.

The term “variant” (also “modified” or mutant“) as used in reference toa variant ICOSL means an ICOSL, such as a mammalian (e.g., human ormurine) ICOSL created by human intervention. The variant ICOSL is apolypeptide having an altered amino acid sequence, relative to anunmodified or wild-type ICOSL. The variant ICOSL is a polypeptide whichdiffers from a wild-type ICOSL isoform sequence by one or more aminoacid substitutions, deletions, additions, or combinations thereof. Forpurposes herein, the variant ICOSL contains at least one affinitymodified domain, whereby one or more of the amino acid differencesoccurs in an IgSF domain (e.g. IgV domain). A variant ICOSL can contain1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid differences,such as amino acid substitutions. A variant ICOSL polypeptide generallyexhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to acorresponding wild-type or unmodified ICOSL, such as to the sequence ofSEQ ID NO:5, a mature sequence thereof or a portion thereof containingthe extracellular domain or an IgSF domain thereof. In some embodiments,a variant ICOSL polypeptide exhibits at least 50%, 60%, 70%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to a corresponding wild-type or unmodified ICOSLcomprising the sequence set forth in SEQ ID NO:32 or SEQ ID NO: 196.Non-naturally occurring amino acids as well as naturally occurring aminoacids are included within the scope of permissible substitutions oradditions. A variant ICOSL is not limited to any particular method ofmaking and includes, for example, de novo chemical synthesis, de novorecombinant DNA techniques, or combinations thereof. A variant ICOSL ofthe invention specifically binds to at least one or more of: CD28, ICOS,or CTLA-4 of a mammalian species . In some embodiments, the alteredamino acid sequence results in an an altered (i.e., increased ordecreased) binding affinity or avidity to ICOS and/or CD28 compared tothe wild-type ICOSL protein. An increase or decrease in binding affinityor avidity can be determined using well known binding assays such asflow cytometry. Larsen et al., American Journal of Transplantation, Vol5: 443-453 (2005). See also, Linsley et al., Immunity, 1: 7930801(1994). An increase in variant ICOSL binding affinity or avidity to ICOSand/or CD28 is to a value at least 5% greater than that of the wild-typeICOSL and in some embodiments, at least 10%, 15%, 20%, 30%, 40%, 50%,100% greater than that of the wild-type ICOSL control value. A decreasein ICOSL binding affinity or avidity to ICOS and/or CD28 is to a valueno greater than 95% of the of the wild-type control values, and in someembodiments no greater than 80%, 70% 60%, 50%, 40%, 30%, 20%, 10%, 5%,or no detectable binding affinity or avidity of the wild-type ICOSand/or CD28 control values. A variant ICOSL is altered in primary aminoacid sequence by substitution, addition, or deletion of amino acidresidues. The term “variant” in the context of variant ICOSL is not beconstrued as imposing any condition for any particular startingcomposition or method by which the variant ICOSL is created. A variantICOSL can, for example, be generated starting from wild type mammalianICOSL sequence information, then modeled in silico for binding to ICOSand/or CD28, and finally recombinantly or chemically synthesized toyield a variant ICOSL of the present invention. In but one alternativeexample, a variant ICOSL can be created by site-directed mutagenesis ofa wild-type ICOSL. Thus, variant ICOSL denotes a composition and notnecessarily a product produced by any given process. A variety oftechniques including recombinant methods, chemical synthesis, orcombinations thereof, may be employed.

The term “wild-type” or “natural” or “native” as used herein is used inconnection with biological materials such as nucleic acid molecules,proteins (e.g., ICOSL), IgSF members, host cells, and the like, refersto those which are found in nature and not modified by humanintervention.

II. VARIANT ICOSL POLYPEPTIDES

Provided herein are variant ICOSL polypeptides that exhibit altered(increased or decreased) binding activity or affinity for one or more ofan ICOSL cognate binding partner. In some embodiments, the ICOSL cognatebinding partner is CD28, ICOS, or CTLA-4. In some embodiments, thevariant ICOSL polypeptide contains one or more amino acidsmodifications, such as one or more substitutions (alternatively,“mutations” or “replacements”), deletions or addition, in animmunoglobulin superfamily (IgSF) domain (IgD) relative to a wild-typeor unmodified ICOSL polypeptide or a portion of a wild-type orunmodified ICOSL containing an immunoglobulin superfamily (IgSF) domainor a specific binding fragment thereof. Thus, a provided variant ICOSLpolypeptide is or comprises a variant IgD (hereinafter called “vIgD”) inwhich the one or more amino acid modifications (e.g. substitutions) isin an IgD.

In some embodiments, the IgD comprises an IgV domain or an IgC (e.g.IgC2) domain or specific binding fragment of the IgV domain or the IgC(e.g. IgC2) domain, or combinations thereof. In some embodiments, theIgD can be an IgV only, the combination of the IgV and IgC, includingthe entire extracellular domain (ECD), or any combination of Ig domainsof ICOSL. Table 2 provides exemplary residues that correspond to IgV orIgC regions of ICOSL. In some embodiments, the variant ICOSL polypeptidecontains an IgV domain or an IgC domain or specific binding fragmentsthereof in which the at least one of the amino acid modifications (e.g.substitutions) is in the IgV domain or IgC domain or a specific bindingfragment thereof. In some embodiments, by virtue of the altered bindingactivity or affinity, the IgV domain or IgC domain is anaffinity-modified IgSF domain.

In some embodiments, the variant is modified in one more IgSF domainsrelative to the sequence of an unmodified ICOSL sequence. In someembodiments, the unmodified ICOSL sequence is a wild-type ICOSL. In someembodiments, the unmodified or wild-type ICOSL has the sequence of anative ICOSL or an ortholog thereof. In some embodiments, the unmodifiedICOSL is or comprises the extracellular domain (ECD) of ICOSL or aportion thereof containing one or more IgSF domain (see Table 2). Insome embodiments, the extracellular domain of an unmodified or wild-typeICOSL polypeptide comprises an IgV domain and an IgC domain or domains.However, the variant ICOSL polypeptide need not comprise both the IgVdomain and the IgC domain or domains. In some embodiments, the variantICOSL polypeptide comprises or consists essentially of the IgV domain ora specific binding fragment thereof. In some embodiments, the variantICOSL polypeptide comprises or consists essentially of the IgC domain orspecific binding fragments thereof. In some embodiments, the variantICOSL is soluble and lacks a transmembrane domain. In some embodiments,the variant ICOSL further comprises a transmembrane domain and, in somecases, also a cytoplasmic domain.

In some embodiments, the wild-type or unmodified ICOSL sequence is amammalian ICOSL sequence. In some embodiments, the wild-type orunmodified ICOSL sequence can be a mammalian ICOSL that includes, but isnot limited to, human, mouse, cynomolgus monkey, or rat. In someembodiments, the wild-type or unmodified ICOSL sequence is human.

In some embodiments, the wild-type or unmodified ICOSL sequence has (i)the sequence of amino acids set forth in SEQ ID NO:5 or a mature formthereof lacking the signal sequence, (ii) a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:5 or themature form thereof, or (iii) is a portion of (i) or (ii) containing anIgV domain or IgC domain or specific binding fragments thereof.

In some embodiments, the wild-type or unmodified ICOSL sequence is orcomprises an extracellular domain of the ICOSL or a portion thereof. Insome embodiments, the unmodified or wild-type ICOSL polypeptidecomprises the amino acid sequence set forth in SEQ ID NO:32, or anortholog thereof. In some cases, the unmodified or wild-type ICOSLpolypeptide can comprise (i) the sequence of amino acids set forth inSEQ ID NO:32, (ii) a sequence of amino acids that has at least about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% sequence identity to SEQ ID NO: 32, or (iii) is a specific bindingfragment of the sequence of(i) or (ii) comprising an IgV domain or anIgC domain.

In some embodiments, the wild-type or unmodified ICOSL polypeptidecomprises an IgV domain or an IgC domain, or a specific binding fragmentthereof. In some embodiments, the IgV domain of the wild-type orunmodified ICOSL polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 196 (corresponding to amino acid residues 19-129 of SEQ IDNO:5), or an ortholog thereof. For example, the IgV domain of theunmodified or wild-type ICOSL polypeptide can contain (i) the sequenceof amino acids set forth in SEQ ID NO: 196, (ii) a sequence of aminoacids that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 196,or (iii) a specific binding fragment of the sequence of amino acids setforth in SEQ ID NO: 196 or a specific binding fragment of a sequence of(i) or (ii). In some embodiments, the wild-type or unmodified IgV domainis capable of binding one or more ICOSL cognate binding proteins, suchas one or more of CD28, ICOS, or CTLA-4.

In some embodiments, the IgC domain of the wild-type or unmodified ICOSLpolypeptide comprises the amino acid sequence set forth as residues141-227 of SEQ ID NO: 5, or an ortholog thereof. For example, the IgCdomain of the unmodified or wild-type ICOSL polypeptide can contain (i)the sequence of amino acids set forth residues 141-227 of SEQ ID NO: 5,(ii) a sequence of amino acids that has at least about 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequenceidentity to residues 141-227 of SEQ ID NO: 5, or (iii) (i) or (ii). Insome embodiments, the wild-type or unmodified IgV domain is capable ofbinding one or more ICOSL cognate binding proteins.

In some embodiments, the wild-type or unmodified ICOSL polypeptidecontains a specific binding fragment of ICOSL, such as a specificbinding fragment of the IgV domain or the IgC domain. In someembodiments the specific binding fragment can bind CD28, ICOS, and/orCTLA-4. The specific binding fragment can have an amino acid length ofat least 50 amino acids, such as at least 60, 70, 80, 90, 100, or 110amino acids. In some embodiments, the specific binding fragment of theIgV domain contains an amino acid sequence that is at least about 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ofthe length of the IgV domain set forth as amino acids 19-129 of SEQ IDNO: 5. In some embodiments, the specific binding fragment of the IgCdomain comprises an amino acid sequence that is at least about 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of thelength of the IgC domain set forth as amino acids 141-227 of SEQ ID NO:5.

In some embodiments, the variant ICOSL polypeptide comprises the ECDdomain or a portion thereof comprising one or more affinity modifiedIgSF domains. In some embodiments, the variant ICOSL polypeptides cancomprise an IgV domain or an IgC domain, in which one or more of theIgSF domains (IgV or IgC) or a specific binding fragment of the IgVdomain or a specific binding fragment of the IgC domain contains the oneor more amino acid modifications (e.g. substitutions). In someembodiments, the variant ICOSL polypeptides can comprise an IgV domainand an IgC domain, or a specific binding fragment of the IgV domain anda specific binding fragment of the IgC domain. In some embodiments, thevariant ICOSL polypeptide comprises a full-length IgV domain. In someembodiments, the variant ICOSL polypeptide comprises a full-length IgCdomain. In some embodiments, the variant ICOSL polypeptide comprises aspecific binding fragment of the IgV domain. In some embodiments, thevariant ICOSL polypeptide comprises a specific binding fragment of theIgC domain. In some embodiments, the variant ICOSL polypeptide comprisesa full-length IgV domain and a full-length IgC domain. In someembodiments, the variant ICOSL polypeptide comprises a full-length IgVdomain and a specific binding fragment of an IgC domain. In someembodiments, the variant ICOSL polypeptide comprises a specific bindingfragment of an IgV domain and a full-length IgC domain. In someembodiments, the variant ICOSL polypeptide comprises a specific bindingfragment of an IgV domain and a specific binding fragment of an IgCdomain.

In any of such embodiments, the one or more amino acid modifications(e.g. substitutions) of the variant ICOSL polypeptides can be located inany one or more of the ICOSL polypeptide domains. For example, in someembodiments, one or more amino acid substitutions are located in theextracellular domain of the variant ICOSL polypeptide. In someembodiments, one or more amino acid substitutions are located in the IgVdomain or specific binding fragment of the IgV domain. In someembodiments, one or more amino acid modifications (e.g. substitutions)are located in the IgC domain or specific binding fragment of the IgCdomain.

Generally, each of the various attributes of polypeptides are separatelydisclosed below (e.g., soluble, secretable and membrane boundpolypeptides, affinity of ICOSL for CD28, ICOS, and CTLA-4, number ofvariations per polypeptide chain, number of linked polypeptide chains,the number and nature of amino acid alterations per variant ICOSL,etc.). However, as will be clear to the skilled artisan, any particularpolypeptide can comprise a combination of these independent attributes.It is understood that reference to amino acids, including to a specificsequence set forth as a SEQ ID NO used to describe domain organizationof an IgSF domain are for illustrative purposes and are not meant tolimit the scope of the embodiments provided. It is understood thatpolypeptides and the description of domains thereof are theoreticallyderived based on homology analysis and alignments with similarmolecules. Thus, the exact locus can vary, and is not necessarily thesame for each protein. Hence, the specific IgSF domain, such as specificIgV domain or IgC domain, can be several amino acids (such as one, two,three or four) longer or shorter.

Further, various embodiments of the invention as discussed below arefrequently provided within the meaning of a defined term as disclosedabove. The embodiments described in a particular definition aretherefore to be interpreted as being incorporated by reference when thedefined term is utilized in discussing the various aspects andattributes described herein. Thus, the headings, the order ofpresentation of the various aspects and embodiments, and the separatedisclosure of each independent attribute is not meant to be a limitationto the scope of the present disclosure.

Exemplary Modifications

Provided herein are variant ICOSL polypeptides containing at least oneaffinity-modified IgSF domain (e.g. IgV or IgC) or a specific bindingfragment thereof in an IgSF domain contained in a wild-type orunmodified ICOSL polypeptide such that the variant ICOSL polypeptideexhibits altered (increased or decreased) binding activity or affinityfor one or more ligands ICOS, CD28, or CTLA-4 compared to a wild-type orunmodified ICOSL polypeptide. In some embodiments, a variant ICOSLpolypeptide has a binding affinity for CD28, ICOS, and/or CTLA-4 thatdiffers from that of a wild-type or unmodified ICOSL polypeptide controlsequence as determined by, for example, solid-phase ELISA immunoassays,flow cytometry or Biacore assays. In some embodiments, the variant ICOSLpolypeptide has an increased binding affinity for CD28, ICOS, and/orCTLA-4. In some embodiments, the variant ICOSL polypeptide has adecreased binding affinity for CD28, ICOS, and/or CTLA-4, relative to awild-type or unmodified ICOSL polypeptide. The CD28, ICOS and/or theCTLA-4 can be a mammalian protein, such as a human protein or a murineprotein.

Binding affinities for each of the cognate binding partners areindependent; that is, in some embodiments, a variant ICOSL polypeptidehas an increased binding affinity for one, two or three of CD28, ICOS,and/or CTLA-4, and a decreased binding affinity for one, two or three ofCD28, ICOS, and CTLA-4, relative to a wild-type or unmodified ICOSLpolypeptide.

In some embodiments, the variant ICOSL polypeptide has an increasedbinding affinity for CD28, relative to a wild-type or unmodified ICOSLpolypeptide. In some embodiments, the variant ICOSL polypeptide has anincreased binding affinity for ICOS, relative to a wild-type orunmodified ICOSL polypeptide. In some embodiments, the variant ICOSLpolypeptide has an increased binding affinity for CTLA-4, relative to awild-type or unmodified ICOSL polypeptide. In some embodiments, thevariant ICOSL polypeptide has a decreased binding affinity for CD28,relative to a wild-type or unmodified ICOSL polypeptide. In someembodiments, the variant ICOSL polypeptide has a decreased bindingaffinity for ICOS, relative to a wild-type or unmodified ICOSLpolypeptide. In some embodiments, the variant ICOSL polypeptide has adecreased binding affinity for CTLA-4, relative to a wild-type orunmodified ICOSL polypeptide.

In some embodiments, the variant ICOSL polypeptide has an increasedbinding affinity for CD28 and ICOS, relative to a wild-type orunmodified ICOSL polypeptide. In some embodiments, the variant ICOSLpolypeptide has an increased binding affinity for CD28 and a decreasedbinding affinity for ICOS, relative to a wild-type or unmodified ICOSLpolypeptide. In some embodiments, the variant ICOSL polypeptide has adecreased binding affinity for CD28 and ICOS, relative to a wild-type orunmodified ICOSL polypeptide. In some embodiments, the variant ICOSLpolypeptide has a decreased binding affinity for CD28 and an increasedbinding affinity for ICOS, relative to a wild-type or unmodified ICOSLpolypeptide.

In some embodiments, the variant ICOSL polypeptide has an increasedbinding affinity for CD28 and CTLA-4, relative to a wild-type orunmodified ICOSL polypeptide. In some embodiments, the variant ICOSLpolypeptide has an increased binding affinity for CD28 and a decreasedbinding affinity for CTLA-4, relative to a wild-type or unmodified ICOSLpolypeptide. In some embodiments, the variant ICOSL polypeptide has adecreased binding affinity for CD28 and CTLA-4, relative to a wild-typeor unmodified ICOSL polypeptide. In some embodiments, the variant ICOSLpolypeptide has a decreased binding affinity for CD28 and an increasedbinding affinity for CTLA-4, relative to a wild-type or unmodified ICOSLpolypeptide.

In some embodiments, the variant ICOSL polypeptide has an increasedbinding affinity for ICOS and CTLA-4, relative to a wild-type orunmodified ICOS polypeptide. In some embodiments, the variant ICOSLpolypeptide has an increased binding affinity for ICOS and a decreasedbinding affinity for CTLA-4, relative to a wild-type or unmodified ICOSLpolypeptide. In some embodiments, the variant ICOSL polypeptide has adecreased binding affinity for ICOS and CTLA-4, relative to a wild-typeor unmodified ICOSL polypeptide. In some embodiments, the variant ICOSLpolypeptide has a decreased binding affinity for ICOS and an increasedbinding affinity for CTLA-4, relative to a wild-type or unmodified ICOSLpolypeptide.

In some embodiments, the variant ICOSL polypeptide has an increasedbinding affinity for CD28, ICOS, and CTLA-4, relative to a wild-type orunmodified ICOSL polypeptide. In some embodiments, the variant ICOSLpolypeptide has an increased binding affinity for CD28 and ICOS, and adecreased binding affinity for CTLA-4, relative to a wild-type orunmodified ICOSL polypeptide. In some embodiments, the variant ICOSLpolypeptide has an increased binding affinity for CD28 and CTLA-4, and adecreased binding affinity for ICOS, relative to a wild-type orunmodified ICOSL polypeptide. In some embodiments, the variant ICOSLpolypeptide has a decreased binding affinity for CD28 and ICOS, and anincreased binding affinity for CTLA-4, relative to a wild-type orunmodified ICOSL polypeptide. In some embodiments, the variant ICOSLpolypeptide has a decreased binding affinity for CD28 and an increasedbinding affinity for ICOS and CTLA-4, relative to a wild-type orunmodified ICOSL polypeptide. In some embodiments, the variant ICOSLpolypeptide has an increased binding affinity for CD28, and a decreasedbinding affinity for ICOS and CTLA-4, relative to a wild-type orunmodified ICOSL polypeptide. In some embodiments, the variant ICOSLpolypeptide has a decreased binding affinity for CD28, CTLA-4, and ICOS,relative to a wild-type or unmodified ICOSL polypeptide. In someembodiments, the variant ICOSL polypeptide has a decreased bindingaffinity for CD28, and an increased binding affinity for ICOS andCTLA-4, relative to a wild-type or unmodified ICOSL polypeptide.

In some embodiments, a variant ICOSL polypeptide with increased orgreater binding affinity to CD28, ICOS, and/or CTLA-4 will have anincrease in binding affinity relative to the wild-type or unmodifiedICOSL polypeptide control of at least about 5%, such as at least about10%, 15%, 20%, 25%, 35%, or 50% for the CD28, ICOS, and/or CTLA-4. Insome embodiments, the increase in binding affinity relative to thewild-type or unmodified ICOSL polypeptide is more than 1.2-fold,1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 30-fold, 40-fold or 50-fold. In such examples,the wild-type or unmodified ICOSL polypeptide has the same sequence asthe variant ICOSL polypeptide except that it does not contain the one ormore amino acid modifications (e.g. substitutions).

In some embodiments, a variant ICOSL polypeptide with reduced ordecreased binding affinity to CD28, ICOS, and/or CTLA-4 will havedecrease in binding affinity relative to the wild-type or unmodifiedICOSL polypeptide control of at least 5%, such as at least about 10%,15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more for the CD28, ICOSL,and/or CTLA-4. In some embodiments, the decrease in binding affinityrelative to the wild-type or unmodified ICOSL polypeptide is more than1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 20-fold, 30-fold 40-fold or 50-fold. In suchexamples, the wild-type or unmodified ICOSL polypeptide has the samesequence as the variant ICOSL polypeptide except that it does notcontain the one or more amino acid modifications, e.g. substitutions.

In some embodiments, the equilibrium dissociation constant (K_(d)) ofany of the foregoing embodiments to CD28, ICOS, and/or CTLA-4 can beless than 1×10⁻⁵ M, 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M or1×10⁻¹¹ M, or 1×10⁻¹² M.

In some embodiments, a variant ICOSL polypeptide has an increased orgreater binding affinity to CD28. In some embodiments, a variant ICOSLpolypeptide with increased or greater binding affinity to CD28 will havean increase in binding affinity relative to the wild-type or unmodifiedICOSL polypeptide control of at least about 25%, such as at least about30%, 40%, 50%, or 60% for CD28. In some embodiments, a variant ICOSLpolypeptide with increased or greater binding affinity to CD28 has anequilibrium dissociation constant (K_(d)) of less than 200 pM, 300 pM,400 pM, 500 pM, or 600 pM for CD28. In some embodiments, the variantpolypeptide specifically binds to the ectodomain of one of ICOS, CD28 orCTLA4 with increased selectivity compared to the unmodified ICOSL. Insome embodiments, the increased selectivity is for CD28. In someembodiments, the increased selectivity comprises a greater ratio ofbinding of the variant ICOSL polypeptide for one cognate binding partnerselected from among ICOS, CD28 and CTLA4 versus another of the cognatebinding partner compared to the ratio of binding of the unmodified ICOSLpolypeptide for the one cognate binding partner versus the another ofthe cognate binding partner. In some embodiments, the ratio is greaterby at least or at least about 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold,5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold or more.

The wild-type or unmodified ICOSL sequence does not necessarily have tobe used as a starting composition to generate variant ICOSL polypeptidesdescribed herein. Therefore, use of the term “modification”, such as“substitution” does not imply that the present embodiments are limitedto a particular method of making variant ICOSL polypeptides. VariantICOSL polypeptides can be made, for example, by de novo peptidesynthesis and thus does not necessarily require a modification, such asa “substitution”, in the sense of altering a codon to encode for themodification, e.g. substitution. This principle also extends to theterms “addition” and “deletion” of an amino acid residue which likewisedo not imply a particular method of making. The means by which thevariant ICOSL polypeptides are designed or created is not limited to anyparticular method. In some embodiments, however, a wild-type orunmodified ICOSL encoding nucleic acid is mutagenized from wild-type orunmodified ICOSL genetic material and screened for desired specificbinding affinity and/or induction of IFN-gamma expression or otherfunctional activity. In some embodiments, a variant ICOSL polypeptide issynthesized de novo utilizing protein or nucleic acid sequencesavailable at any number of publicly available databases and thensubsequently screened. The National Center for Biotechnology Informationprovides such information and its website is publicly accessible via theinternet as is the UniProtKB database as discussed previously.

Unless stated otherwise, as indicated throughout the present disclosure,the amino acid substitution(s) are designated by amino acid positionnumber corresponding to the numbering of positions of the unmodified ECDsequence set forth in SEQ ID NO:32 or, where applicable, the unmodifiedIgV sequence set forth in SEQ ID NO:196 (containing residues 19-129 ofSEQ ID NO:32) as follows:

(SEQ ID NO: 32) DTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKITENPVSTGEKNAAT (SEQ ID NO: 196)DTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQ SLGFQEVLSVE

It is within the level of a skilled artisan to identify thecorresponding position of a modification, e.g. amino acid substitution,in an ICOSL polypeptide, including portion thereof containing an IgSFdomain (e.g. IgV) thereof, such as by alignment of a reference sequencewith SEQ ID NO:32 or SEQ ID NO:196. In the listing of modificationsthroughout this disclosure, the amino acid position is indicated in themiddle, with the corresponding unmodified (e.g. wild-type) amino acidlisted before the number and the identified variant amino acidsubstitution listed after the number. If the modification is a deletionof the position a “del” is indicated and if the modification is aninsertion at the position an “ins” is indicated.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution in a wild-type or unmodified ICOSLsequence. The one or more amino acid modification, e.g. substitution canbe in the ectodomain (extracellular domain) of the wild-type orunmodified ICOSL sequence. In some embodiments, the one or more aminoacid modification, e.g. substitution is in the IgV domain or specificbinding fragment thereof. In some embodiments, the one or more aminoacid modification, e.g. substitution is in the IgC domain or specificbinding fragment thereof. In some embodiments of the variant ICOSLpolypeptide, some of the one or more amino acid modification, e.g.substitution is in the IgV domain or a specific binding fragmentthereof, and some of the one or more amino acid modification, e.g.substitution are in the IgC domain or a specific binding fragmentthereof.

In some embodiments, the variant ICOSL polypeptide has up to 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidmodification(s), e.g. substitution. The modification, e.g. substitutioncan be in the IgV domain or the IgC domain. In some embodiments, thevariant ICOSL polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions in theIgV domain or specific binding fragment thereof. In some embodiments,the variant ICOSL polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions inthe IgC domain or specific binding fragment thereof. In someembodiments, the variant ICOSL polypeptide has at least about 85%, 86%,86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity with the wild-type or unmodified ICOSL polypeptide orspecific binding fragment thereof, such as with the amino acid sequenceof SEQ ID NO: 32 or 196.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution in an unmodified ICOSL or specificbinding fragment there of corresponding to position(s) 10, 11, 13, 16,18, 20, 25, 27, 30, 33, 37, 42, 43, 47, 52, 54, 57, 61, 62, 67, 71, 72,74, 77, 78, 75, 80, 84, 89, 90, 92, 93, 94, 96, 97, 98, 99, 100, 102,103, 107, 109, 110, 111, 113, 115, 116, 117, 119, 120, 121, 122, 126,129, 130, 132, 133, 135, 138, 139, 140, 142, 143, 144, 146, 151, 152,153, 154, 155, 156, 158, 161, 166, 168, 172, 173, 175, 190, 192, 193,194, 198, 201, 203, 207, 208, 210, 212, 217, 218, 220, 221, 224, 225, or227 with reference to numbering of SEQ ID NO:32. In some embodiments,such variant ICOSL polypeptides exhibit altered binding affinity to oneor more of CD28, ICOS, and/or CTLA-4 compared to the wild-type orunmodified ICOSL polypeptide. For example, in some embodiments, thevariant ICOSL polypeptide exhibits increased binding affinity to CD28,ICOS, and/or CTLA-4 compared to a wild-type or unmodified ICOSLpolypeptide. In some embodiments, the variant ICOSL polypeptide exhibitsdecreased binding affinity to CD28, ICOS, or CTLA-4 compared to awild-type or unmodified ICOSL polypeptide.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution selected from M10V, M10I, V11E,S13G, E16V, S18R, A20V, 525G, F27S, F27C, N30D, Y33del, Q37R, K42E,Y47H, T43A, N52H, N52D, N52Q, N52S, N52Y, N52K, S54A, S54P, N57D, N57Y,R61S, R61C, Y62F, L67P, A71T, G72R, L74Q, R75Q, D77G, F78L, L80P, N84Q,D89G, E90A, K92R, F93L, H94E, H94D, L96F, L961, V97A, L98F, S99G, Q100R,Q100K, Q100P, L102R, G103E, V107A, V1071, S109G, 5109N, V110D, V110N,V110A, E111del, T113E, H115R, H115Q, V116A, A117T, N119Q, F1201, F1205,S121G, V122A, V122M, S126T, S126R, H129P, 5130G,5132F, Q133H, E135K,F138L, T1395, C140D, C140del, S142F, I143V, I143T, N144D, Y146C, V151A,Y152C, Y152H, W153R, I154F, N155H, N155Q, K156M, D158G, L161P, L161M,L166Q, N168Q, F1725, L1735, M175T, T1905, T190A, S192G, V193M, N194D,C198R, N201S, L203P, L203F, N207Q, L208P, V210A, S212G, D217V, I218T,I218N, E220G, R221G, R221I, I224V, T225A, N227K or a conservative aminoacid modification, e.g. substitution thereof. A conservative amino acidmodification, e.g. substitution is any amino acid that falls in the sameclass of amino acids as the substituted amino acids, other than thewild-type or unmodified amino acid. The classes of amino acids arealiphatic (glycine, alanine, valine, leucine, and isoleucine), hydroxylor sulfur-containing (serine, cysteine, threonine, and methionine),cyclic (proline), aromatic (phenylalanine, tyrosine, tryptophan), basic(histidine, lysine, and arginine), and acidic/amide (aspartate,glutamate, asparagine, and glutamine).

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution selected from M10V, M10I, V11E,S13G, E16V, S18R, A20V, S25G, F27S, F27C, N30D, Y33del, Q37R, K42E,T43A, Y47H, N52H, N52D, N52Q, N52S, N52Y, N52K, S54A, S54P, N57D, N57Y,R61S, R61C, Y62F, L67P, A71T, G72R, L74Q, R75Q, D77G, F78L, L80P, N84Q,D89G, E90A, K92R, F93L, H94E, H94D, L96F, L961, V97A, L98F, S99G, Q100R,Q100K, Q100P, G103E, L102R, V107A, V1071, S109G, S109N, V110D, V110N,V110A, E111del, T113E, H115R, H115Q, V116A, A117T, N119Q, F1201, F120S,S121G, V122A, V122M, S126T, S126R, H129P, S130G,S132F, Q133H, E135K,F138L, T139S, C140D, C140del, S142F, I143V, I143T, N144D, Y146C, V151A,Y152C, Y152H,W153R, I154F, N155H, N155Q, K156M, D158G, L161P, L161M,L166Q, N168Q, F172S, L173S, M175T, T190A, T190S, S192G, V193M, N194D,C198R, N201S, L203P, L203F, N207Q, L208P, V210A, S212G, D217V, I218T,1218N, E220G, R221G, R221I, I224V, T225A, N227K.

In some embodiments, the one or more amino acid modification, e.g.substitution is N52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P,N52S/Y146C/Y152C, N52H/C198R, N52H/C140D/T225A, N52H/C198R/T225A,N52H/K92R, N52H/S99G, N57Y/Q100P, N52S/G103E, N52S/S130G/Y152C,N52S/Y152C, N52S/C198R, N52Y/N57Y/Y152C, N52Y/N57Y/ H129P/C198R,N52H/L161P/C198R, N52S/T113E, N52D/S54P, N52K/L208P, N52S/Y152H,N52D/V151A, N52H/I143T, N52S/L80P, F120S/Y152H/N201S, N52S/R75Q/L203P,N52S/D158G, N52D/Q133H, N52S/N57Y/H94D/L96F/L98F/Q100R,N52S/N57Y/H94D/L96F/L98F/Q100R/G103E/F120S, N52H/F78L/Q100R,N52H/N57Y/Q100R/V110D, N52H/N57Y/R75Q/Q100R/V110D, N52H/N57Y/Q100R,N52H/N57Y/L74Q/Q100R/V110D, N52H/Q100R, N52H/S121G,A20V/N52H/N57Y/Q100R/S109G, N52H/N57Y/Q100P,N52H/N57Y/R61S/Q100R/V110D/L173S, N52H/N57Y/Q100R/V122A,N52H/N57Y/Q100R/F172S, N52H/N57Y, N52S/F120S, N52S/V97A, N52S/G72R,N52S/A71T/A117T, N52S/E220G, Y47H/N52S/V107A/F120S,N52H/N57Y/Q100R/V110D/S132F/M175T,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R,Q37R/N52H/N57Y/Q100R/V110N/S142F/C198R/D217V/R221G,N52H/N57Y/Q100R/V110D/C198R,N52H/N57Y/Q100R/V110D/V116A/L161M/F172S/S192G/C198R,F27S/N52H/N57Y/V110N, N52S/H94E/L96I/S109N/L166Q,S18R/N52S/F93L/I143V/R221G, A20T/N52D/Y146C/Q164L,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T,N52S/H94E/L96I/V122M, N52H/N57Y/H94E/L96I/F120I/S126T/W153R/I218N,M10V/S18R/N30D/N52S/S126R/T139S/L203F, S25G/N30D/N52S/F120S/N227K,N30D/N52S/L67P/Q100K/D217G/R221K/T225S,N52H/N57Y/Q100R/V110D/A117T/T190S/C198R,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R,N52H/N57Y/V110A/C198R/R221I,M10I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M,C198R,N52H/N57Y/R61C/Y62F/Q100R/V110N/F120S/C198R,N52H/N57Y/Q100R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/N144D/F172S/C198R, N52S/H94E/L98F/Q100R,N52S/E90A, N30D/K42E/N52S, N52S/F120S/I143V/I224V,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/S54P, T38P/N52S/N57D,N52H/C140del/T225A, N52H/F78L/Q100R/C198R, N52H/N57Y/R75Q/Q100P/V110D,N52H/N57Y/L74Q/V110D/S192G, N52H/S121G/C198R, N52S/F120S/N227K,N52S/A71T/A117T/T190A/C198R, T43A/N52H/N57Y/L74Q/D89G/V110D/F172S,N52H/N57Y/Q100R/V110D/S132F/M175T,N52H/N57Y/Q100R/V107I/V110D/I154F/C198R/R221G, Q100R, F138L/L203P,N57Y/F138L/L203P, N57Y/Q100R/C198R, N57Y/F138L/L203P, Q100R/F138L,L203P, N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R,N52H/N57Y/Q100R/L102R/H115R/F172S/C198R, N52H/V122A/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/N194D, N52H/N57Y/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/H115R, N52H/N57Y/Q100R/H115R,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/F172S, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/E90A/H115R, N30D/K42E N52S/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N52S/H115R/F120S/I143V/C198R, N52S/H115R/F172S/C198R,N52H/N57Y/Q100P/C198R, N52H/N57Y/Q100P H115R/F172S/C198R,N52H/N57Y/Q100P/F172S/C198R, N52H/N57Y/Q100P/H115R,N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R, N52H/Q100R/H115R/F172S,N52H/Q100R/F172S/C198R, N52H/Q100R/H115Q/F172S/C198R, N52H/N57Y/Q100R/F172S/C198R, N52Q/N207Q, N168Q/N207Q, N52Q/N168Q, N84Q/N207Q,N155Q/N207Q, N119Q/N168Q , N119Q/N207Q, N119Q/N155Q, N52Q/N84Q,N52Q/N119Q, N84Q/N119Q, N52Q/N84Q/N168Q, N52Q/N84Q/N207Q,N84Q/N155Q/N168Q, N84Q/N168Q/N207Q, N84Q/N155H/N207Q, N155Q/N168Q/N207Q,N119Q/N155Q/N168Q, N119Q/N168Q/N207Q, N84Q/N119Q/N207Q,N119Q/N155H/N207Q, N84Q/N119Q/N155Q, N52Q/N119Q/N155Q, N52H/N84Q/N119Q,N52H/N84Q, N52H/N84Q/N168Q, N52H/N84Q/N207Q, N52H/N84Q/N168Q/N207Q,N52Q/N84Q/N155Q, N52Q/N84Q/ N168Q, N52Q/N84Q/N155Q/N168Q,N52Q/N84Q/N119Q/N168Q, N84Q/N119Q/N155Q/N168Q, N84Q/N155Q/N168Q/N207Q,N84Q/N119Q/N155Q/N207Q, N52Q/N84Q/N119Q/N207Q, N52Q/N84Q/N119Q/N155Q,N52Q/N84Q/N119Q/N155Q/N207Q or, N84Q/N119Q/N155Q/N168Q/N207Q.

In some embodiments, the variant ICOSL polypeptide comprises any of themutations listed in Table 1. Table 1 also provides exemplary sequencesby reference to SEQ ID NO for the extracellular domain (ECD) or IgVdomain of wild-type ICOSL or exemplary variant ICOSL polypeptides. Asindicated, the exact locus or residues corresponding to a given domaincan vary, such as depending on the methods used to identify or classifythe domain. Also, in some cases, adjacent N- and/or C-terminal aminoacids of a given domain (e.g. IgV) also can be included in a sequence ofa variant IgSF polypeptide, such as to ensure proper folding of thedomain when expressed. Thus, it is understood that the exemplificationof the SEQ ID NOSs in Table 1 is not to be construed as limiting. Forexample, the particular domain, such as the ECD domain, of a variantICOSL polypeptide can be several amino acids longer or shorter, such as1-10, e.g. 1, 2, 3, 4, 5, 6 or 7 amino acids longer or shorter, than thesequence of amino acids set forth in the respective SEQ ID NO.

In some embodiments, the variant ICOSL polypeptide comprises any of themutations listed in Table 1. In some embodiments, the variant ICOSLpolypeptide comprises any of the extracellular domain (ECD) sequenceslisted in Table 1 (i.e., any one of SEQ ID NOS: 109-142, 239, 280-325,364-381, 387-424, 427-433, 435-470). In some embodiments, the variantICOSL polypeptide comprises a polypeptide sequence that exhibits atleast 90% identity, at least 91% identity, at least 92% identity, atleast 93% identity, at least 94% identity, at least 95% identity, suchas at least 96% identity, 97% identity, 98% identity, or 99% identity toany of the extracellular domain (ECD) sequences listed in Table 1 (i.e.,any one of SEQ ID NOS: 109-142, 239, 280-325, 364-381, 387-424, 427-433,435-470) and contains the amino acid modification(s), e.g.substitution(s) not present in the wild-type or unmodified ICOSL. Insome embodiments, the variant ICOSL polypeptide comprises a specificbinding fragment of any of the extracellular domain (ECD) sequenceslisted in Table 1 (i.e., any one of SEQ ID NOS: 109-142, 239, 280-325,364-381, 387-424, 427-433, 435-470) and contains the amino acidmodification(s), e.g. substitution (s) not present in the wild-type orunmodified ICOSL. In some embodiments, the variant ICOSL polypeptidecomprises any of the IgV sequences listed in Table 1 (i.e., any one ofSEQ ID NOS: 197-199, 201-208, 210, 212, 240, 326-340, 382-386, 425-426,and 434). In some embodiments, the variant ICOSL polypeptide comprises apolypeptide sequence that exhibits at least 90% identity, at least 91%identity, at least 92% identity, at least 93% identity, at least 94%identity, at least 95% identity, such as at least 96% identity, 97%identity, 98% identity, or 99% identity to any of the IgV sequenceslisted in Table 1 (i.e., any one of SEQ ID NOS: 197-199, 201-208, 210,212, 240, 326-340, 382-386, 425-426, and 434) and contains the aminoacid modification(s), e.g. substitution(s) not present in the wild-typeor unmodified ICOSL. In some embodiments, the variant ICOSL polypeptidecomprises a specific binding fragment of any of the IgV sequences listedin Table 1 (i.e., any one of SEQ ID NOS: 197-199, 201-208, 210, 212,240, 326-340, 382-386, 425-426, and 434) and contains the amino acidsubstitution(s) not present in the wild-type or unmodified ICOSL.Mutations designated with an “X” indicate the designated positioncontains a Q or the wildtype residue set forth in the correspondingposition of SEQ ID NO: 32.

TABLE 1 Exemplary variant ICOSL polypeptides ECD SEQ IgV ID SEQ IDMutation(s) NO NO Wild-type 32 196 N52S 109 197 N52H 110 198 N52D 111199 N52Y/N57Y/F138L/L203P 112 N52H/N57Y/Q100P 113 201 N52S/Y146C/Y152C114 N52H/C198R 115 N52H/C140D/T225A 116 N52H/C198R/T225A 117 N52H/K92R118 202 N52H/S99G 119 203 N52Y 120 204 N57Y 121 205 N57Y/Q100P 122 206N52S/S130G/Y152C 123 N52S/Y152C 124 N52S/C198R 125 N52Y/N57Y/Y152C 126N52Y/N57Y/H129P/C198R 127 N52H/L161P/C198R 128 N52S/T113E 129 S54A 130207 N52D/S54P 131 208 N52K/L208P 132 N52S/Y152H 133 N52D/V151A 134N52H/I143T 135 N52S/L80P 136 210 F120S/Y152H/N201S 137 N52S/R75Q/L203P138 N52S/D158G 139 N52D/Q133H 140 N52S/N57Y/H94D/L96F/L98F/Q100R 141 212N52S/N57Y/H94D/L96F/L98F/Q100R/G103E/F120S 142 N52S/G103E 239 240N52H/F78L/Q100R 280 326 N52H/N57Y/Q100R/V110D 281 327N52H/N57Y/R75Q/Q100R/V110D 282 328 N52H/N57Y/Q100R 283 329N52H/N57Y/L74Q/Q100R/V110D 284 330 N52H/Q100R 285 331 N52H/S121G 286A20V/N52H/N57Y/Q100R/S109G 287 332 N52H/N57Y/Q100P 288 333N52H/N57Y/R61S/Q100R/V110D/L173S 289 N52H/N57Y/Q100R/V122A 290N52H/N57Y/Q100R/F172S 291 N52H/N57Y 292 334 N52S/F120S 293 N52S/V97A 294335 N52S/G72R 295 336 N52S/A71T/A117T 296 N52S/E220G 297Y47H/N52S/V107A/F120S 298 N52H/N57Y/Q100R/V110D/S132F/M175T 299E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R 300Q37R/N52H/N57Y/Q100R/V110N/S142F/C198R/D217V/R221G 301N52H/N57Y/Q100R/V110D/C198R 302N52H/N57Y/Q100R/V110D/V116A/L161M/F172S/S192G/C198R 303F27S/N52H/N57Y/V110N 304 337 N52S/H94E/L96I/S109N/L166Q 305S18R/N52S/F93L/I143V/R221G 306 A20T/N52D/Y146C/Q164L 307V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T 308N52S/H94E/L96I/V122M 309 N52H/N57Y/H94E/L96I/F120I/S126T/W153R/I218N 310M10V/S18R/N30D/N52S/S126R/T139S/L203F 311 S25G/N30D/N52S/F120S/N227K 312N30D/N52S/L67P/Q100K/D217G/R221K/T225S 313N52H/N57Y/Q100R/V110D/A117T/T190S/C198R 314N52H/N57Y/Q100R/V110D/F172S/C198R 315S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R 316N52H/N57Y/V110A/C198R/R221I 317M10I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M/C198R 318N52H/N57Y/R61C/Y62F/Q100R/V110N/F120S/C198R 319N52H/N57Y/Q100R/V110D/H115R/C198R 320N52H/N57Y/Q100R/V110D/N144D/F172S/C198R 321 N52S/H94E/L98F/Q100R 322 338N52S/E90A 323 339 N30D/K42E/N52S 324 340 N52S/F120S/I143V/I224V 325N52H/N57Y/Q100R/V110D/C198R/S212G 364 N52H/N57Y/Q100R/C198R 365N52S/N194D 366 N52H/N57Y/Q100R/L102R/V110D/H115R/C198R 367 N52S/S54P 368382 T38P/N52S/N57D 369 383 E111del 370 384 Y33del 371 385N52H/C140del/T225A 372 N52H/F78L/Q100R/C198R 373N52H/N57Y/R75Q/Q100P/V110D 374 386 N52H/N57Y/L74Q/V110D/S192G 375N52H/S121G/C198R 376 N52S/F120S/N227K 377 N52S/A71T/A117T/T190A/C198R378 T43A/N52H/N57Y/L74Q/D89G/V110D/F172S 379N52H/N57Y/Q100R/V110D/S132F/M175T 380N52H/N57Y/Q100R/V107I/V110D/I154F/C198R/R221G 381 N84Q 387 425 N119Q 388N168Q 389 N207Q 390 N52Q/N207X 391 N168X/N207X 392 N52Q/N168Q 393N84Q/N207Q 394 N155Q/N207Q 395 N119Q/N168Q 396 N119Q/N207Q 397N119Q/N155X 398 N52Q/N84Q 399 426 N52Q/N119Q 400 N84Q/N119Q 401N52Q/N84Q/N168Q 402 N52Q/N84Q/N207Q 403 N84Q/N155Q/N168Q 404N84Q/N168Q/N207Q 405 N84Q/N155H/N207Q 406 N155Q/N168Q/N207Q 407 N119QN155Q/N168Q 408 N119Q/N168Q/N207Q 409 N84Q/N119Q/N207Q 410N119Q/N155H/N207Q 411 N84Q/N119Q/N155Q 412 N52Q/N119Q/N155Q 413N52H/N84Q/N119Q 414 N52H/N84Q/N168X/N207X 415 N52Q/N84Q/N155X/N168X 416N52Q/N84Q/N119Q/N168Q 417 N84Q/N119Q/N155Q/N168Q 418N84Q/N155Q/N168Q/N207Q 419 N84Q/N119Q/N155Q/N207Q 420N52Q/N84Q/N119Q/N207Q 421 N52Q/N84Q/N119Q/N155Q 422N52Q/N84Q/N119Q/N155Q/N207Q 423 N84Q/N119Q/N155Q/N168Q/N207Q 424 Q100R427 434 F138L/L203P 428 N52Y/F138L/L203P 429 N57Y/Q100R/C198R 430N57Y/F138L/L203P 431 Q100R/F138L 432 L203P 433N52H/N57Y/Q100R/H115R/C198R 435 N52H/N57Y/Q100R/F172S/C198R 436N52H/N57Y/Q100R/H115R/F172S/C198R 437N52H/N57Y/Q100R/H115R/I143V/F172S/C198R 438N52H/N57Y/Q100R/L102R/H115R/F172S/C198R 439 N52H/V122A/F172S/C198R 440N52H/N57Y/Q100R/H115R/F172S/N194D 441 N52H/N57Y/H115R/F172S/C198R 442N52H/N57Y/Q100R/H115R/C198R 443 N52H/N57Y/H115R 444N52H/N57Y/Q100R/H115R 445 N52H/N57Y/Q100R/H115R/F172S/I224V 446N52H/N57Y/Q100R/H115R/F172S 447 N52H/N57Y/Q100R/F172S 448N52H/Q100R/H115R/I143T/F172S 449 N52H/N57Y/Q100P/H115R/F172S 450N52Y/N57Y/Q100P/F172S 451 E16V/N52H/N57Y/Q100R/V110D/H115R/C198R 452E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R 453N52S/E90A/H115R 454 N30D/K42E N52S/H115R 455N30D/K42E/N52S/H115R/C198R/R221I 456 N30D/K42E/N52S/H115R/C198R 457N30D/K42E/N52S/H115R/F172S/N194D 458 N52S/H115R/F120S/I143V/C198R 459N52S/H115R/F172S/C198R 460 N52H/N57Y/Q100P/C198R 461 N52H/N57Y/Q100PH115R/F172S/C198R 462 N52H/N57Y/Q100P/F172S/C198R 463N52H/N57Y/Q100P/H115R 464 N52H/N57Y/Q100P/H115R/C198R 465N52H/Q100R/C198R 466 N52H/Q100R/H115R/F172S 467N52H/Q100R/H115X/F172S/C198R 468 N52H/Q100R/H115R/F172S/C198R 469N52H/N57Y/Q100R/F172S/C198R 470

In some embodiments, the variant ICOSL polypeptide exhibits increasedaffinity for the ectodomain of CD28 compared to the wild-type orunmodified ICOSL polypeptide, such as comprising the sequence set forthin SEQ ID NO:32 or 196. In some embodiments, the ICOSL polypeptideexhibits increased affinity for the ectodomain of ICOS compared to thewild-type or unmodified ICOSL, such as comprising the sequence set forthin SEQ ID NO:32 or 196. In some embodiments, the ICOSL polypeptideexhibits increased affinity for the ectodomain of CD28 and theectodomain of ICOS compared to the wild-type or unmodified ICOSL, suchas comprising the sequence set forth in SEQ ID NO:32 or 196.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution corresponding to position(s) 52, 54or 57. In some embodiments, the variant ICOSL polypeptide has one ormore amino acid modification, e.g. substitution selected from N52H,N52D, N52Q, N52S, N52Y, N52K, S54A, S54P, or N57Y or a conservativeamino acid modification, e.g. substitution thereof. In some embodiments,the variant ICOSL polypeptide has one or more amino acid modification,e.g. substitution selected from N52H, N52D, N52S, N52K or N57Y or aconservative amino acid modification, e.g. substitution thereof.

In some embodiments, the variant ICOSL polypeptide can contain one ormore further amino acid modification, e.g. substitution in addition toan amino acid modification, e.g. substitution at a positioncorresponding to position 52, 54 or 57. In some embodiments, the one ormore further amino acid modification, e.g. substitution is at a positioncorresponding to 10, 11, 13, 16, 18, 20, 25, 27, 30, 37, 42, 43, 47, 52,54, 57, 61, 62, 67, 71, 72, 74, 75, 77, 78, 80, 84, 89, 90, 92, 93, 94,96, 97, 98, 99, 100, 102, 103, 107, 109, 110, 113, 115, 116, 117, 119,120, 121, 122, 126, 129, 130, 132, 133, 135, 138, 139, 140, 142, 143,144, 146, 151, 152, 153, 154, 155, 156, 158, 161, 166, 168, 172, 173,175, 190, 192, 193, 194, 198, 201, 203, 207, 208, 210, 212, 217, 218,220, 221, 224, 225 or 227. In some embodiments, the variant ICOSLcontains one or more further amino acid modification, e.g. substitutionselected from M10V, M10I, V11E, S13G, E16V, S18R, A20V, S25G, F27S,F27C, N30D, Y33del, Q37R, K42E, T43A, Y47H, N52H, N52D, N52S, N52Y,N52K, N52Q, S54A, S54P, N57D, N57Y, R61S, R61C, Y62F, L67P, A71T, G72R,L74Q, R75Q, D77G, F78L, L80P, N84Q, D89G, E90A, K92R, F93L, H94E, H94D,L96F, L961, V97A, L98F, S99G, Q100R, Q100K, Q100P, L102R, G103E, V107A,V1071, S109G, S109N, V110D, V110N, V110A, E111del, T113E, H115R, H115Q,V116A, A117T, N119Q, F120I, F120S,S121G, V122A, V122M, S126T, S126R,H129P, S130G,S132F, Q133H, E135K, F138L, T139S, C140del, S142F,I143V,I143T, N144D, Y146C, V151A, Y152C, Y152H,W153R, I154F, K156M, D158G,L161P, L161M, L166Q, N168Q, F172S, L173S, M175T, T190A, T190S, S192G,V193M, N194D, C198R, N201S, L203P, L203F, N207Q, L208P, V210A, S212G,D217V, I218T, 1218N, E220G, R221G, R221I, I224V, T225A, N227K, or aconservative amino acid substitution thereof.

In some embodiments of any one of the variant ICOSL polypeptidesdescribed above, the variant ICOSL polypeptide further comprises one ormore amino acid deletions corresponding to positions 140 of SEQ ID NO:32.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution selected fromN52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P, N52S/Y146C/Y152C, N52H/C198R,N52H/C140del/T225A, N52H/C198R/T225A, N52H/K92R, N57Y/Q100P, N52S/C198R,N52Y/N57Y/Y152C, N52Y/N57Y/H129P/C198R, N52H/L161P/C198R, N52S/T113E,N52S/S54P, N52K/L208P, N52S/Y152H, N52H/I143T, N52S/R75Q/L203P,N52S/D158G, N52D/Q133H, N52H/ N57Y/Q100R/V110D/C198R/S212G,N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/S54P, T38P/N52S/N57D,N52H/C140del/T225A, N52H/F78L/Q100R/C198R, N52H/N57Y/R75Q/Q100P/V110D,N52H/N57Y/L74Q/V110D/S192G, N52H/S121G/C198R, N52S/F120S/N227K,N52S/A71T/A117T/T190A/C198R, T43A/N52H/N57Y/L74Q/D89G/V110D/F172S,N52H/N57Y/Q100R/V110D/S132F/M175T,N52H/N57Y/Q100R/V1071/V110D/1154F/C198R/R221G, N52Q/N207Q, N52Q/N168Q,N52Q/N84Q, N52Q/N119Q, N52Q/N84Q/N168Q, N52Q/N84Q/N207Q,N52Q/N119Q/N155Q, N52H/N84Q/N119Q, N52H/N84Q, N52H/N84Q/N168Q,N52H/N84Q/N207Q, N52H/N84Q/N168Q/N207Q, N52Q/N84Q/N155Q,N52Q/N84Q/N168Q, N52Q/N84Q/N155Q/N168Q, N52Q/N84Q/N119Q/N168Q,N52Q/N84Q/N119Q/N207Q, N52Q/N84Q/N119Q/N155Q,N52Q/N84Q/N119Q/N155Q/N207Q, N52Y/F138L/L203P, N57Y/Q100R/C198R,N57Y/F138L/L203P, N52H/N57Y/Q100R/H115R/C198R,N52H/N57Y/Q100R/F172S/C198R, N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/1143V/F172S/C198R,N52H/N57Y/Q100R/L102R/H115R/F172S/C198R, N52H/V122A/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/N194D, N52H/N57Y/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/H115R, N52H/N57Y/Q100R/H115R,N52H/N57Y/Q100R/H115R/F172S/1224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/F172S, N52H/Q100R/H115R/1143T/F172S,N52H/N57Y/Q100P/H115R/F172S, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/E90A/H115R, N30D/K42E/N52S/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N52S/H115R/F120S/1143V/C198R, N52S/H115R/F172S/C198R,N52H/N57Y/Q100P/C198R, N52H/N57Y/Q100P H115R/F172S/C198R,N52H/N57Y/Q100P/F172S/C198R, N52H/N57Y/Q100P/H115R,N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R, N52H/Q100R/H115R/F172S,N52H/Q100R/ F172S/C198R, N52H/Q100R/H115R/F172S/C198R, orN52H/N57Y/Q100R/F172S/C198R.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution selected fromN52H/N57Y/Q100R/C198R, N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S,N52Y/N57Y/F138L/L203P,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52H/N57Y/Q100R, N52H/Q100R,N52H/N57Y/Q100R/V110D/C198R/S212G,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/V152C/K156M/C198R, N30D/K42E/N52S,N52S/F120S/I143V/I224V, N52S/E90A, N52H/N57Y/V110A/C198R/R221I,N52H/N57Y/Q100P, or N52S/N194D.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution selected fromN52H/N57Y/Q100R/F172S, N52H/Q100R, N52H/N57Y/Q100R/C198R. In someembodiments, the variant ICOSL polypeptide has one or more amino acidmodification, e.g. substitution selected fromE16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R, N52H/N57Y/Q100R, andN52H/N57Y/Q100P.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution selected fromN52H/N57Y/F138L/L203P, N52H/N57Y/Q100P, N52H/K92R, N52H/C140del/T225A,N52H/C198R/T225A, N52H/K92R, N57Y/Q100P, N52Y/N57Y/H129P/C198R,N52H/L161P/C198R, N52K/L208P or N52H/I143T.

In some embodiments, the variant ICOSL polypeptide exhibits increasedbinding affinity for binding one of the ectodomains of CD28 or ICOS andexhibits decreased binding affinity for binding to the other of theectodomains of CD28 or ICOS compared to the wild-type or unmodifiedICOSL polypeptide, such as comprising the sequence set forth in SEQ IDNO:32 or 196.

In some embodiments, the variant ICOSL polypeptide exhibits increasedbinding affinity for ICOS and exhibits decreased binding affinity forCD28. In some embodiments, the one or more further amino acidsubstitution is at a position corresponding to 52, 57, 80 100, 130, 152,161 or 198. In some embodiments, the variant ICOSL contains one or moreamino acid substitutions selected from N52S, N52H, N52Y, N52H, N57Y,L80P, Q100P Q100R, Q100K, V110D, S130G, Y152C, L161P, L161M, C198R,R221G, or a conservative amino acid substitution thereof. In someembodiments, the variant ICOSL polypeptide has one or more amino acidsubstitutions selected from N57Y/Q100P, N52S/S130G/Y152C, N52S/Y152C,N52Y/N57Y/Y152C, N52H/L161P/C198R, N52H/L161P/C198R, N52S/L80P,A20V/N52H/N57Y/Q100R/S109G, N52H/N57Y/R61S/Q100R/V110D/L173S,N52H/N57Y/Q100R/V107I/V110D/S132F/I154F/C198R/R221G,Q37R/N52H/N57Y/Q100R/V110N/S142F/C198R/D217V/R221G,N52H/N57Y/Q100R/V110D/C198R, F27S/N52H/N57Y/V110N,S18R/N52S/F93L/I143V/R221G, A20T/N52D/Y146C/Q164L,N52H/N57Y/H94E/L96I/F120I/S126T/W153R/I218N,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R,M10I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M/C198R.

In some embodiments, the variant ICOSL polypeptide exhibits increasedbinding affinity for CD28 and exhibits decreased binding affinity forICOS. In some embodiments, the one or more amino acid substitution is ata position corresponding to 52, 75 or 203. In some embodiments, thevariant ICOSL contains one or more amino acid substitution selected fromN52S, R75Q, L203F, or L203P. In some embodiments, the variant ICOSLpolypeptide has amino acid substitutions N52S/R75Q/L203P.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution in an unmodified ICOSL or specificbinding fragment there of corresponding to position(s) 16, 30, 42, 52,57, 90, 100, 102, 110, 115, 120, 122, 138, 143, 152, 156, 172, 194, 198,203, 221, or 224 with reference to numbering of SEQ ID NO:32. In someembodiments, the variant ICOSL polypeptide has one or more amino acidmodification, e.g. substitution selected from E16V, N30D, K42E, N52H,N52Y, N52S, N57Y, E90A, Q100R, Q100P, L102R, V110D, H115R, F120S, V122A,F138L, I143V, I143T, H152C, K156M, F172S, N194D, C198R, L203P, R221I, orI224V. In some embodiments, the variant ICOSL polypeptide has one ormore amino acid modification, e.g. substitution in an unmodified ICOSLor specific binding fragment there of corresponding to position(s) 115,172, or 198 with reference to numbering of SEQ ID NO:32. In someembodiments, the variant ICOSL polypeptide has one or more amino acidmodification, e.g. substitution selected from H115R, F172S or C198R. Insome embodiments, the one or more amino acid modification, e.g.substitution is N52H/N57Y/Q100R/H115R/C198R,N52H/N57Y/Q100R/F172S/C198R, N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R,N52H/N57Y/Q100R/L102R/H115R/F172S/C198R, N52H/V122A/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/N194D, N52H/N57Y/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/H115R, N52H/N57Y/Q100R/H115R,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/F172S, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/E90A/H115R, N30D/K42E N52S/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N52S/H115R/F120S/I143V/C198R, N52S/H115R/F172S/C198R,N52H/N57Y/Q100P/C198R, N52H/N57Y/Q100P H115R/F172S/C198R,N52H/N57Y/Q100P/F172S/C198R, N52H/N57Y/Q100P/H115R,N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R, N52H/Q100R/H115R/F172S,N52H/Q100R/F172S/C198R, N52H/Q100R/H115R/F172S/C198R or N52H/N57Y/Q100R/F172S/C198R. In some embodiments, the variant ICOSL polypeptidesexhibit potentially enhanced protein solubility or enhanced proteinexpression (‘solubility mutations’) compared to the wild-type orunmodified ICOSL polypeptide.

In some embodiments, the variant ICOSL polypeptide comprises any of theextracellular domain (ECD) sequences set forth in SEQ ID NOS: 435-470.In some embodiments, the variant ICOSL polypeptide comprises apolypeptide sequence that exhibits at least 90% identity, at least 91%identity, at least 92% identity, at least 93% identity, at least 94%identity, at least 95% identity, such as at least 96% identity, 97%identity, 98% identity, or 99% identity to any of the extracellulardomain (ECD) set forth in SEQ ID NOS: 435-470 and contains the aminoacid modification(s), e.g. substitution(s) not present in the wild-typeor unmodified ICOSL. In some embodiments, the variant ICOSL polypeptidecomprises a specific binding fragment of any of the extracellular domain(ECD) sequences set forth in SEQ ID NOS: 435-470 and contains the aminoacid modification(s), e.g. substitution (s) not present in the wild-typeor unmodified ICOSL.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution in an unmodified ICOSL or specificbinding fragment there of corresponding to position(s)52, 57, 100, 138,198, or 203 with reference to numbering of SEQ ID NO:32. In someembodiments, the variant ICOSL polypeptide has one or more amino acidmodification, e.g. substitution selected from N52H, N52Y, N57Y, Q100R,Q100P, F138L, C198R, or L203P. In some embodiments, the one or moreamino acid modification, e.g. substitution is Q100R, F138L/L203P,N52Y/F138L/L203P, N57Y/Q100R/C198R, N57Y/F138L/L203P, N52H, N57Y,N57Y/Q100P, Q100R/F138L, or L203P.

In some embodiments, the variant ICOSL polypeptide comprises any of theextracellular domain (ECD) sequences set forth in SEQ ID NOS: 427-433.In some embodiments, the variant ICOSL polypeptide comprises apolypeptide sequence that exhibits at least 90% identity, at least 91%identity, at least 92% identity, at least 93% identity, at least 94%identity, at least 95% identity, such as at least 96% identity, 97%identity, 98% identity, or 99% identity to any of the extracellulardomain (ECD) set forth in SEQ ID NOS: 427-433 and contains the aminoacid modification(s), e.g. substitution(s) not present in the wild-typeor unmodified ICOSL. In some embodiments, the variant ICOSL polypeptidecomprises a specific binding fragment of any of the extracellular domain(ECD) sequences set forth in SEQ ID NOS: 427-433 and contains the aminoacid modification(s), e.g. substitution (s) not present in the wild-typeor unmodified ICOSL. In some embodiments, the variant ICOSL polypeptidecomprises the IgV sequence set forth in SEQ ID NO: 434. In someembodiments, the variant ICOSL polypeptide comprises a polypeptidesequence that exhibits at least 90% identity, at least 91% identity, atleast 92% identity, at least 93% identity, at least 94% identity, atleast 95% identity, such as at least 96% identity, 97% identity, 98%identity, or 99% identity to the IgV sequence set forth in SEQ ID NO:434 and contains the amino acid modification(s), e.g. substitution(s)not present in the wild-type or unmodified ICOSL. In some embodiments,the variant ICOSL polypeptide comprises a specific binding fragment ofthe IgV sequence set forth in SEQ ID NO: 434 and contains the amino acidsubstitution(s) not present in the wild-type or unmodified ICOSL.

In some embodiments, the variant ICOSL polypeptide has one or more aminoacid modification, e.g. substitution in an unmodified ICOSL or specificbinding fragment there of corresponding to position(s) 52, 84, 91, 119,155, 168, 207 with reference to numbering of SEQ ID NO:32. In someembodiments, the variant ICOSL polypeptide has one or more amino acidmodification, e.g. substitution selected from A91S, N52H, N52Q, N84Q,N119Q, N155H, N155Q, N168Q, N207Q. In some embodiments, the one or moreamino acid modification, e.g. substitution is N84Q, N119Q, N168Q, N207Q,N52Q, N52Q/N207Q, N168Q/N207Q, N52Q/N168Q, N84Q/N207Q, N155Q/N207Q,N119Q/N168Q , N119Q/N207Q, N119Q/N155Q, N52Q/N84Q, N52Q/N119Q,N84Q/N119Q, N52Q/N84Q/N168Q, N52Q/N84Q/N207Q, N84Q/N155Q/N168Q,N84Q/N168Q/N207Q, N84Q/N155H/N207Q, N155Q/N168Q/N207Q, N119QN155Q/N168Q, N119Q/N168Q/N207Q, N84Q/N119Q/N207Q, N119Q/N155H/N207Q,N84Q/N119Q/N155Q, N52Q/N119Q/N155Q, N52H/N84Q/N119Q, N52H/N84Q,N52H/N84Q/N168Q, N52H/N84Q/N207Q, N52H/N84Q/N168Q/N207Q,N52Q/N84Q/N155Q, N52Q/N84Q/ N168Q, N52Q/N84Q/N155Q/N168Q,N52Q/N84Q/N119Q/N168Q, N84Q/N119Q/N155Q/N168Q, N84Q/N155Q/N168Q/N207Q,N84Q/N119Q/N155Q/N207Q, N52Q/N84Q/N119Q/N207Q, N52Q/N84Q/N119Q/N155Q,N52Q/N84Q/N119Q/N155Q/N207Q, N84Q/N119Q/N155Q/N168Q/N207Q orA91S/N119Q/N168Q/N207Q. In some embodiments, the variant ICOSLpolypeptides exhibit potentially reduced glycosylation compared to thewild-type or unmodified ICOSL polypeptide.

In some embodiments, the variant ICOSL polypeptide comprises any of theextracellular domain (ECD) sequences set forth in SEQ ID NOS: 387-424,427-433, 435-470. In some embodiments, the variant ICOSL polypeptidecomprises a polypeptide sequence that exhibits at least 90% identity, atleast 91% identity, at least 92% identity, at least 93% identity, atleast 94% identity, at least 95% identity, such as at least 96%identity, 97% identity, 98% identity, or 99% identity to any of theextracellular domain (ECD) set forth in SEQ ID NOS: 387-424, 427-433,435-470 and contains the amino acid modification(s), e.g.substitution(s) not present in the wild-type or unmodified ICOSL. Insome embodiments, the variant ICOSL polypeptide comprises a specificbinding fragment of any of the extracellular domain (ECD) sequences setforth in SEQ ID NOS: 387-424, 427-433, 435-470 and contains the aminoacid modification(s), e.g. substitution (s) not present in the wild-typeor unmodified ICOSL. In some embodiments, the variant ICOSL polypeptidecomprises any of the IgV sequences set forth in SEQ ID NOS: 425-426. Insome embodiments, the variant ICOSL polypeptide comprises a polypeptidesequence that exhibits at least 90% identity, at least 91% identity, atleast 92% identity, at least 93% identity, at least 94% identity, atleast 95% identity, such as at least 96% identity, 97% identity, 98%identity, or 99% identity to any of the IgV sequences set forth in SEQID NO: 425-426 and contains the amino acid modification(s), e.g.substitution(s) not present in the wild-type or unmodified ICOSL. Insome embodiments, the variant ICOSL polypeptide comprises a specificbinding fragment of any of the IgV sequences set forth in SEQ ID NO:425-426 and contains the amino acid substitution(s) not present in thewild-type or unmodified ICOSL.

III. FORMATS OF VARIANT POLYPEPTIDES

The immunomodulatory polypeptide comprising a variant ICOSL providedherein in which is contained a vIgD can be formatted in a variety ofways, including as a soluble protein, membrane bound protein, secretedprotein, conjugate or fusion or for expression by an infectious agent.In some embodiments, the particular format can be chosen for the desiredtherapeutic application. In some cases, an immunomodulatory polypeptidecomprising a variant ICOSL polypeptide is provided in a format toantagonize or block activity of its cognate binding partner, e.g. CD28.In some embodiments, antagonism of CD28 may be useful to treatinflammation or autoimmunity. In some cases, an immunomodulatorypolypeptide comprising a variant ICOSL polypeptide is provided in aformat to agonize or stimulate activity of its cognate binding partner,e.g. CD28. In some embodiments, agonism of CD28 may be useful fortreating oncology indications. A skilled artisan can readily determinethe activity of a particular format, such as for antagonizing oragonizing one or more specific cognate binding partner. Exemplarymethods for assessing such activities are provided herein, including inthe examples.

In some aspects, provided are immunomodulatory proteins comprising avIgD of ICOSL in which such proteins are soluble, e.g. fused to an Fcchain. In some aspects, one or more additional IgSF domain, such as oneor more additional vIgD, may be linked to a vIgD of ICOSL as providedherein (hereinafter called a “stack” or “stacked” immunomodulatoryprotein). In some embodiments, the modular format of the providedimmunomodulatory proteins provides flexibility for engineering orgenerating immunomodulatory proteins for modulating activity of multiplecounterstrucutres (multiple cognate binding partners). In someembodiments, such “stack” molecules can be provided in a soluble formator, in some cases, may be provided as membrane bound or secretedproteins. In some embodiments, a variant ICOSL immunomodulatory proteinis provided as a conjugate in which is contained a vIgD of ICOSL linked,directly or indirectly, to a targeting agent or moiety, e.g. to anantibody or other binding molecules that specifically binds to a ligand,e.g. an antigen, for example, for targeting or localizing the vIgD to aspecific environment or cell, such as when administered to a subject. Insome embodiments, the targeting agent, e.g. antibody or other bindingmolecule, binds to a tumor antigen, thereby localizing the variant ICOSLcontaining the vIgD to the tumor microenvironment, for example, tomodulate activity of tumor infiltrating lymphocytes (TILs) specific tothe tumor microenvironment.

In some embodiments, provided immunomodulatory proteins are expressed incells and provided as part of an engineered cellular therapy (ECT). Insome embodiments, the variant ICOSL polypeptide is expressed in a cell,such as an immune cell (e.g. T cell or antigen presenting cell), inmembrane-bound form, thereby providing a transmembrane immunomodulatoryprotein (hereinafter also called a “TIP”). In some embodiments,depending on the cognate binding partner recognized by the TIP,engineered cells expressing a TIP can agonize a cognate binding partnerby providing a costimulatory signal, either positive to negative, toother engineered cells and/or to endogenous T cells. In some aspects,the variant ICOSL polypeptide is expressed in a cell, such as an immunecell (e.g. T cell or antigen presenting cell), in secretable form tothereby produce a secreted or soluble form of the variant ICOSLpolypeptide (hereinafter also called a “SIP”), such as when the cellsare administered to a subject. In some aspects, a SIP can antagonize acognate binding partner in the environment (e.g. tumor microenvironment)in which it is secreted. In some embodiments, a variant ICOSLpolypeptide is expressed in an infectious agent (e.g. viral or bacterialagent) which, upon administration to a subject, is able to infect a cellin vivo, such as an immune cell (e.g. T cell or antigen presenting cell)or tumor, for delivery or expression of the variant polypeptide as a TIPor a SIP in the cell.

In some embodiments, a soluble immunomodulatory polypeptide, such as avariant ICOSL containing a vIgD, can be encapsulated within a liposomewhich itself can be conjugated to any one of or any combination of theprovided conjugates (e.g., a targeting moiety). In some embodiments, thesoluble or membrane bound immunomodulatory polypeptides of the inventionare deglycosylated. In more specific embodiments, the variant ICOSLsequence is deglycosylated. In even more specific embodiments, the IgVand/or IgC (e.g. IgC2) domain or domains of the variant ICOSL isdeglycosylated.

Non-limiting examples of provided formats are described in FIGS. 13A-13Cand further described below.

A. Soluble Protein

In some embodiments, the immunomodulatory protein containing a variantICOSL polypeptide is a soluble protein. Those of skill will appreciatethat cell surface proteins typically have an intracellular,transmembrane, and extracellular domain (ECD) and that a soluble form ofsuch proteins can be made using the extracellular domain or animmunologically active subsequence thereof. Thus, in some embodiments,the immunomodulatory protein containing a variant ICOSL polypeptidelacks a transmembrane domain or a portion of the transmembrane domain.In some embodiments, the immunomodulatory protein containing a variantICOSL lacks the intracellular (cytoplasmic) domain or a portion of theintracellular domain. In some embodiments, the immunomodulatory proteincontaining the variant ICOSL polypeptide only contains the vIgD portioncontaining the ECD domain or a portion thereof containing an IgV domainand/or IgC (e.g. IgC2) domain or domains or specific binding fragmentsthereof containing the amino acid modification(s).

In some embodiments, an immunomodulatory polypeptide comprising avariant ICOSL can include one or more variant ICOSL polypeptides of theinvention. In some embodiments a polypeptide of the invention willcomprise exactly 1, 2, 3, 4, 5 variant ICOSL sequences. In someembodiments, at least two of the variant ICOSL sequences are identicalvariant ICOSL sequences.

In some embodiments, the provided immunomodulatory polypeptide comprisestwo or more vIgD sequences of ICOSL. Multiple variant ICOSL polypeptideswithin the polypeptide chain can be identical (i.e., the same species)to each other or be non-identical (i.e., different species) variantICOSL sequences. In addition to single polypeptide chain embodiments, insome embodiments two, three, four, or more of the polypeptides of theinvention can be covalently or non-covalently attached to each other.Thus, monomeric, dimeric, and higher order (e.g., 3, 4, 5, or more)multimeric proteins are provided herein. For example, in someembodiments exactly two polypeptides of the invention can be covalentlyor non-covalently attached to each other to form a dimer. In someembodiments, attachment is made via interchain cysteine disulfide bonds.Compositions comprising two or more polypeptides of the invention can beof an identical species or substantially identical species ofpolypeptide (e.g., a homodimer) or of non-identical species ofpolypeptides (e.g., a heterodimer). A composition having a plurality oflinked polypeptides of the invention can, as noted above, have one ormore identical or non-identical variant ICOSL polypeptides of theinvention in each polypeptide chain.

In some embodiments, the immunomodulatory protein comprises a variantICOSL polypeptide attached to an immunoglobulin Fc (yielding an“immunomodulatory Fc fusion,” such as a “ICOSL-Fc variant fusion,” alsotermed a ICOSL vIgD-Fc fusion). In some embodiments, the attachment ofthe variant ICOSL polypeptide is at the N-terminus of the Fc. In someembodiments, the attachment of the variant ICOSL polypeptide is at theC-terminus of the Fc. In some embodiments, two or more ICOSL variantpolypeptides (the same or different) are independently attached at theN-terminus and at the C-terminus.

In some embodiments, the Fc is murine or human Fc. In some embodiments,the Fc is a mammalian or human IgG1, IgG2, IgG3, or IgG4 Fc regions. Insome embodiments, the Fc is derived from IgG1, such as human IgG1. Insome embodiments, the Fc comprises the amino acid sequence set forth inSEQ ID NO: 226 or a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to SEQ ID NO: 226.

In some embodiments, the Fc region contains one more modifications toalter (e.g. reduce) one or more of its normal functions. In general, theFc region is responsible for effector functions, such ascomplement-dependent cytotoxicity (CDC) and antibody-dependent cellcytotoxicity (ADCC), in addition to the antigen-binding capacity, whichis the main function of immunoglobulins. Additionally, the FcRn sequencepresent in the Fc region plays the role of regulating the IgG level inserum by increasing the in vivo half-life by conjugation to an in vivoFcRn receptor. In some embodiments, such functions can be reduced oraltered in an Fc for use with the provided Fc fusion proteins.

In some embodiments, one or more amino acid modifications may beintroduced into the Fc region of a ICOSL-Fc variant fusion providedherein, thereby generating an Fc region variant. In some embodiments,the Fc region variant has decreased effector function. There are manyexamples of changes or mutations to Fc sequences that can alter effectorfunction. For example, WO 00/42072, WO2006019447, WO2012125850,WO2015/107026, US2016/0017041 and Shields et al. J Biol. Chem. 9(2):6591-6604 (2001) describe exemplary Fc variants with improved ordiminished binding to FcRs. The contents of those publications arespecifically incorporated herein by reference.

In some embodiments, the provided variant ICOSL-Fc fusions comprise anFc region that exhibits reduced effector functions, which makes it adesirable candidate for applications in which the half-life of theICOSL-Fc variant fusion in vivo is important yet certain effectorfunctions (such as CDC and ADCC) are unnecessary or deleterious. Invitro and/or in vivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theICOSL-Fc variant fusion lacks FCγR binding (hence likely lacking ADCCactivity), but retains FcRn binding ability. The primary cells formediating ADCC, NK cells, express FCγRIII only, whereas monocytesexpress FCγRI, FCγRII and FCγRIII FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays toassess ADCC activity of a molecule of interest is described in U.S. Pat.No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al.,J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assaymethods may be employed (see, for example, ACTI™ non-radioactivecytotoxicity assay for flow cytometry (CellTechnology, Inc. MountainView, Calif.; and CytoTox 96™ non-radioactive cytotoxicity assay(Promega, Madison, Wis.). Useful effector cells for such assays includeperipheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in an animal model such as thatdisclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).C1q binding assays may also be carried out to confirm that the ICOSL-Fcvariant fusion is unable to bind C1q and hence lacks CDC activity. See,e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. Toassess complement activation, a CDC assay may be performed (see, forexample, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996);Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivoclearance/half life determinations can also be performed using methodsknown in the art (see, e.g., Petkova, S. B. et al., Int'l Immunol.18(12):1759-1769 (2006)).

ICOSL-Fc variant fusions with reduced effector function include thosewith substitution of one or more of Fc region residues 238, 265, 269,270, 297, 327 and 329 by EU numbering (U.S. Pat. No. 6,737,056). Such Fcmutants include Fc mutants with substitutions at two or more of aminoacid positions 265, 269, 270, 297 and 327 by EU numbering, including theso-called “DANA” Fc mutant with substitution of residues 265 and 297 toalanine (U.S. Pat. No. 7,332,581).

In some embodiments, the Fc region of ICOSL-Fc variant fusions has an Fcregion in which any one or more of amino acids at positions 234, 235,236, 237, 238, 239, 270, 297, 298, 325, and 329 (indicated by EUnumbering) are substituted with different amino acids compared to thenative Fc region. Such alterations of Fc region are not limited to theabove-described alterations, and include, for example, alterations suchas deglycosylated chains (N297A and N297Q), IgG1-N297G,IgG1-L234A/L235A, IgG1-L234A/L235E/G237A, IgG1-A325A/A330S/P331S,IgGl-C226S/C229S, IgG1-C226S/C229S/E233P/L234V/L235A, IgG1-E233P/L234V/L235A/G236del/S267K, IgG1-L234F/L235E/P331S,IgG1-S267E/L328F, IgG2-V234A/G237A, IgG2-H268Q/V309L/A330S/A331S,IgG4-L235A/G237A/E318A, and IgG4-L236E described in Current Opinion inBiotechnology (2009) 20 (6), 685-691; alterations such as G236R/L328R,L235G/G236R, N325A/L328R, and N325LL328R described in WO 2008/092117;amino acid insertions at positions 233, 234, 235, and 237 (indicated byEU numbering); and alterations at the sites described in WO 2000/042072.

Certain Fc variants with improved or diminished binding to FcRs aredescribed. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312,WO2006019447 and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In some embodiments, there is provided a ICOSL-Fc variant fusioncomprising a variant Fc region comprising one or more amino acidsubstitutions which increase half-life and/or improve binding to theneonatal Fc receptor (FcRn). Antibodies with increased half-lives andimproved binding to FcRn are described in US2005/0014934A1 (Hinton etal.) or WO2015107026. Those antibodies comprise an Fc region with one ormore substitutions therein which improve binding of the Fc region toFcRn. Such Fc variants include those with substitutions at one or moreof Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312,317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 by EUnumbering, e.g., substitution of Fc region residue 434 (U.S. Pat. No.7,371,826).

In some embodiments, the Fc region of a ICOSL-Fc variant fusioncomprises one or more amino acid substitution E356D and M358L. In someembodiments, the Fc region of a ICOSL-Fc variant fusion comprises one ormore amino acid substitutions C220S, C226S, C229S. In some embodiments,the Fc region of a ICOSL variant fusion comprises one or more amino acidsubstitutions R292C and V302C. See also Duncan & Winter, Nature322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; andWO 94/29351 concerning other examples of Fc region variants.

In some embodiments, alterations are made in the Fc region that resultin diminished C1q binding and/or Complement Dependent Cytotoxicity(CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, andIdusogie et al., J. Immunol. 164: 4178-4184 (2000).

In some embodiments, there is provided a ICOSL-Fc variant fusioncomprising a variant Fc region comprising one or more amino acidmodifications, wherein the variant Fc region is derived from IgG1, suchas human IgG1. In some embodiments, the variant Fc region is derivedfrom the amino acid sequence set forth in SEQ ID NO: 226. In someembodiments, the Fc contains at least one amino acid substitution thatis N82G by numbering of SEQ ID NO: 226 (corresponding to N297G by EUnumbering). In some embodiments, the Fc further contains at least oneamino acid substitution that is R77C or V87C by numbering of SEQ ID NO:226 (corresponding to R292C or V302C by EU numbering). In someembodiments, the variant Fc region further comprises a C5S amino acidmodification by numbering of SEQ ID NO: 226 (corresponding to C220S byEU numbering). For example, in some embodiments, the variant Fc regioncomprises the following amino acid modifications: N82G and one or moreof the following amino acid modifications C5S, R77C or V87C withreference to SEQ ID NO:226.

In some embodiments, there is provided a ICOSL-Fc variant fusioncomprising a variant Fc region in which the variant Fc comprises thesequence of amino acids set forth in any of SEQ ID NOS:474, 476, 477,478 or 507 or a sequence of amino acids that exhibits at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to any of SEQ ID NOS: 474, 476, 477, 478 or 507.

In some embodiments, the Fc is derived from IgG2, such as human IgG2. Insome embodiments, the Fc comprises the amino acid sequence set forth inSEQ ID NO: 227 or a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to SEQ ID NO: 227.

In some embodiments, the Fc comprises the amino acid sequence set forthin SEQ ID NO: 505 or a sequence of amino acids that exhibits at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to SEQ ID NO: 505. In some embodiments,the IgG4 Fc is a stabilized Fc in which the CH3 domain of human IgG4 issubstituted with the CH3 domain of human IgG1 and which exhibitsinhibited aggregate formation, an antibody in which the CH3 and CH2domains of human IgG4 are substituted with the CH3 and CH2 domains ofhuman IgG1, respectively, or an antibody in which arginine at position409 indicated in the EU index proposed by Kabat et al. of human IgG4 issubstituted with lysine and which exhibits inhibited aggregate formation(see e.g. U.S. Pat. No. 8,911,726). In some embodiments, the Fc is anIgG4 containing the S228P mutation, which has been shown to preventrecombination between a therapeutic antibody and an endogenous IgG4 byFab-arm exchange (see e.g. Labrijin et al. (2009) Nat. Biotechnol.,27(8)767-71.) In some embodiments, the Fc comprises the amino acidsequence set forth in SEQ ID NO: 506 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 506.

In some embodiments, the variant ICOSL polypeptide is directly linked tothe Fc sequence. In some embodiments, the variant ICOSL polypeptide isindirectly linked to the Fc sequence, such as via a linker. In someembodiments, one or more “peptide linkers” link the variant ICOSLpolypeptide and the Fc domain. In some embodiments, a peptide linker canbe a single amino acid residue or greater in length. In someembodiments, the peptide linker has at least one amino acid residue butis no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1 amino acid residues in length. In some embodiments, thelinker is (in one-letter amino acid code): GGGGS (“4GS”) or multimers ofthe 4GS linker, such as repeats of 2, 3, 4, or 5 4GS linkers.

In some embodiments, the variant ICOSL-Fc fusion protein is a dimerformed by two variant ICOSL Fc polypeptides linked to an Fc domain. Insome specific embodiments, identical or substantially identical species(allowing for 3 or fewer N-terminus or C-terminus amino acid sequencedifferences) of ICOSL-Fc variant fusion polypeptides will be dimerizedto create a homodimer. In some embodiments, the dimer is a homodimer inwhich the two variant ICOSL Fc polypeptides are the same. Alternatively,different species of ICOSL-Fc variant fusion polypeptides can bedimerized to yield a heterodimer. Thus, in some embodiments, the dimeris a heterodimer in which the two variant ICOSL Fc polypeptides aredifferent.

Also provided are nucleic acid molecules encoding the variant ICOSL-Fcfusion protein. In some embodiments, for production of an Fc fusionprotein, a nucleic acid molecule encoding a variant ICOSL-Fc fusionprotein is inserted into an appropriate expression vector. The resultingvariant ICOSL-Fc fusion protein can be expressed in host cellstransformed with the expression where assembly between Fc domains occursby interchain disulfide bonds formed between the Fc moieties to yielddimeric, such as divalent, variant ICOSL-Fc fusion proteins.

The resulting Fc fusion proteins can be easily purified by affinitychromatography over Protein A or Protein G columns. For the generationof heterodimers, additional steps for purification can be necessary. Forexample, where two nucleic acids encoding different variant ICOSLpolypeptides are transformed into cells, the formation of heterodimersmust be biochemically achieved since variant ICOSL molecules carryingthe Fc-domain will be expressed as disulfide-linked homodimers as well.Thus, homodimers can be reduced under conditions that favor thedisruption of interchain disulfides, but do no effect intra-chaindisulfides. In some cases, different variant-ICOSL Fc monomers are mixedin equimolar amounts and oxidized to form a mixture of homo- andheterodimers. The components of this mixture are separated bychromatographic techniques. Alternatively, the formation of this type ofheterodimer can be biased by genetically engineering and expressing Fcfusion molecules that contain a variant ICOSL polypeptide usingknob-into-hole methods described below.

B. Stack Molecules with Additional IgSF Domains

In some embodiments, the immunomodulatory proteins can contain any ofthe variant ICOSL polypeptides provided herein linked, directly orindirectly, to one or more other immunoglobulin superfamily (IgSF)domain (“stacked” immunomodulatory protein construct and also called a“Type II” immunomodulatory protein). In some aspects, this can createunique multi-domain immunomodulatory proteins that bind two or more,such as three or more, cognate binding partners, thereby providing amulti-targeting modulation of the immune synapse.

In some embodiments, an immunomodulatory protein comprises a combination(a “non-wild-type combination”) and/or arrangement (a “non-wild typearrangement” or “non-wild-type permutation”) of a variant ICOSL domainwith one or more other affinity modified and/or non-affinity modifiedIgSF domain sequences of another IgSF family member (e.g. a mammalianIgSF family member) that are not found in wild-type IgSF family members.In some embodiments, the immunomodulatory protein contains 2, 3, 4, 5 or6 immunoglobulin superfamily (IgSF) domains, where at least one of theIgSF domain is a variant ICOSL IgSF domain (vIgD of ICOSL) according tothe provided description.

In some embodiments, the sequences of the additional IgSF domains can bea modified IgSF domain that contains one or more amino acidmodifications, e.g. substitutions, compared to a wildtype or unmodifiedIgSF domain. In some embodiments, the IgSF domain can be non-affinitymodified (e.g., wild-type) or have been affinity modified. In someembodiments, the unmodified or wild-type IgSF domain can be from mouse,rat, cynomolgus monkey, or human origin, or combinations thereof. Insome embodiments, the additional IgSF domains can be an IgSF domain ofan IgSF family member set forth in Table 2. In some embodiments, theadditional IgSF domain can be an affinity-modified IgSF domaincontaining one or more amino acid modifications, e.g. substitutions,compared to an IgSF domain contained in an IgSF family member set forthin Table 2.

In some embodiments, the additional IgSF domain is an affinity ornon-affinity modified IgSF domain contained in an IgSF family member ofa family selected from Signal-Regulatory Protein (SIRP) Family,Triggering Receptor Expressed On Myeloid Cells Like (TREML) Family,Carcinoembryonic Antigen-related Cell Adhesion Molecule (CEACAM) Family,Sialic Acid Binding Ig-Like Lectin (SIGLEC) Family, Butyrophilin Family,B7 family, CD28 family, V-set and Immunoglobulin Domain Containing(VSIG) family, V-set transmembrane Domain (VSTM) family, MajorHistocompatibility Complex (MHC) family, Signaling lymphocyticactivation molecule (SLAM) family, Leukocyte immunoglobulin-likereceptor (LIR), Nectin (Nec) family, Nectin-like (NECL) family,Poliovirus receptor related (PVR) family, Natural cytotoxicitytriggering receptor (NCR) family, T cell immunoglobulin and mucin (TIM)family or Killer-cell immunoglobulin-like receptors (KIR) family. Insome embodiments, the additional IgSF domains are independently derivedfrom an IgSF protein selected from the group consisting of CD80(B7-1),CD86(B7-2), CD274 (PD-L1, B7-H1), PDCD1LG2(PD-L2, CD273), ICOSLG(B7RP1,CD275, ICOSL, B7-H2), CD276(B7-H3), VTCN1(B7-H4), CD28, CTLA4,PDCD1(PD-1), ICOS, BTLA(CD272), CD4, CD8A(CD8-alpha), CD8B(CD8-beta),LAG3, HAVCR2(TIM-3), CEACAM1, TIGIT, PVR(CD155), PVRL2(CD112), CD226,CD2, CD160, CD200, CD200R1(CD200R), and NC R3 (NKp30).

The first column of Table 2 provides the name and, optionally, the nameof some possible synonyms for that particular IgSF member. The secondcolumn provides the protein identifier of the UniProtKB database, apublicly available database accessible via the internet at uniprot.orgor, in some cases, the GenBank Number. The Universal Protein Resource(UniProt) is a comprehensive resource for protein sequence andannotation data. The UniProt databases include the UniProt Knowledgebase(UniProtKB). UniProt is a collaboration between the EuropeanBioinformatics Institute (EMBL-EBI), the SIB Swiss Institute ofBioinformatics and the Protein Information Resource (PIR) and supportedmainly by a grant from the U.S. National Institutes of Health (NIH).GenBank is the NIH genetic sequence database, an annotated collection ofall publicly available DNA sequences (Nucleic Acids Research, 2013January; 41(D1):D36-42). The third column provides the region where theindicated IgSF domain is located. The region is specified as a rangewhere the domain is inclusive of the residues defining the range. Column3 also indicates the IgSF domain class for the specified IgSF region.Colum 4 provides the region where the indicated additional domains arelocated (signal peptide, S; extracellular domain, E; transmembranedomain, T; cytoplasmic domain, C). It is understood that description ofdomains can vary depending on the methods used to identify or classifythe domain, and may be identified differently from different sources.The description of residues corresponding to a domain in Table 2 is forexemplification only and can be several amino acids (such as one, two,three or four) longer or shorter. Column 5 indicates for some of thelisted IgSF members, some of its cognate cell surface binding partners.

TABLE 2 IgSF members according to the present disclosure. IgSF MemberAmino Acid Sequence Cognate Cell (SEQ ID NO) IgSF UniProtKB IgSF RegionSurface Precursor Member Protein & Domain Other Binding (mature(Synonyms) Identifier Class Domains Partners residues) Mature ECD CD80NP_005182.1 35-135, 35-138 S: 1-34, CD28, CTLA4, SEQ ID SEQ ID SEQ ID(B7-1) P33681 or 37-138 IgV, E: 35-242, PD-L1 NO: 1 NO: 253 NO: 28145-230 or T: 243-263, (35-288) 154-232 IgC C: 264-288 CD86 P42081.233-131 IgV, S: 1-23, CD28, CTLA4 SEQ ID SEQ ID SEQ ID (B7-2) 150-225IgC2 E: 24-247, NO: 2 NO: 254 NO: 29 T: 248-268, (24-329) C: 269-329CD274 Q9NZQ7.1 24-130 IgV, S: 1-18, PD-1, B7-1 SEQ ID SEQ ID SEQ ID(PD-L1, 133-225 IgC2 E: 19-238, NO: 3 NO: 255 NO: 30 B7-H1) T: 239-259,(19-290) C: 260-290 PDCD1LG2 Q9BQ51.2 21-118 IgV, S: 1-19, PD-1, RGMbSEQ ID SEQ ID SEQ ID (PD-L2, 122-203 IgC2 E: 20-220, NO: 4 NO: 256 NO:31 CD273) T: 221-241, (20-273) C: 242-273 ICOSLG O75144.2 19-129 IgV, S:1-18, ICOS, CD28, SEQ ID SEQ ID SEQ ID (B7RP1, 141-227 IgC2 E: 19-256,CTLA4 NO: 5 NO: 257 NO: 32 CD275, T: 257-277, (19-302) ICOSL, C: 278-302B7-H2) CD276 Q5ZPR3.1 29-139 IgV, S: 1-28, SEQ ID SEQ ID SEQ ID (B7-H3)145-238 IgC2, E: 29-466, NO: 6 NO: 258 NO: 33 243-357 IgV, T: 467-487,(29-534) 367-453 IgC C: 488-534 VTCN1 Q7Z7D3.1 35-146 IgV, S: 1-24, SEQID SEQ ID SEQ ID (B7-H4) 153-241 IgV E: 25-259, NO: 7 NO: 259 NO: 34 T:260-280, (25-282) C: 281-282 CD28 P10747.1 28-137 IgV S: 1-18, B7-1,B7-2, SEQ ID SEQ ID SEQ ID E: 19-152, B7RP1 NO: 8 NO: 260 NO: 35 T:153-179, (19-220) C: 180-220 CTLA4 P16410.3 39-140 IgV S: 1-35, B7-1,B7-2, SEQ ID SEQ ID SEQ ID E: 36-161, B7RP1 NO: 9 NO: 261 NO: 36 T:162-182, (36-223) C: 183-223 PDCD1 Q15116.3 35-145 IgV S: 1-20, PD-L1,PD-L2 SEQ ID SEQ ID SEQ ID (PD-1) E: 21-170, NO: 10 NO: 262 NO: 37 T:171-191, (21-288) C: 192-288 ICOS Q9Y6W8.1 30-132 IgV S: 1-20, B7RP1 SEQID SEQ ID SEQ ID E: 21-140, NO: 11 NO: 263 NO: 38 T: 141-161, (21-199)C: 162-199 BTLA Q7Z6A9.3 31-132 IgV S: 1-30, HVEM SEQ ID SEQ ID SEQ ID(CD272) E: 31-157, NO: 12 NO: 264 NO: 39 T: 158-178, (31-289) C: 179-289CD4 P01730.1 26-125 IgV, S: 1-25, MHC class II SEQ ID SEQ ID SEQ ID126-203 IgC2, E: 26-396, NO: 13 NO: 265 NO: 40 204-317 IgC2, T: 397-418,(26-458) 317-389 IgC2 C: 419-458 CD8A P01732.1 22-135 IgV S: 1-21, MHCclass I SEQ ID SEQ ID SEQ ID (CD8- E: 22-182, NO: 14 NO: 266 NO: 41alpha) T: 183-203, (22-235) C: 204-235 CD8B P10966.1 22-132 IgV S: 1-21,MHC class I SEQ ID SEQ ID SEQ ID (CD8- E: 22-170, NO: 15 NO: 267 NO: 42beta) T: 171-191, (22-210) C: 192-210 LAG3 P18627.5 37-167 IgV, S: 1-28,MHC class II SEQ ID SEQ ID SEQ ID 168-252 IgC2, E: 29-450, NO: 16 NO:268 NO: 43 265-343 IgC2, T: 451-471, (29-525) 349-419 IgC2 C: 472-525HAVCR2 Q8TDQ0.3 22-124 IgV S: 1-21, CEACAM-1, SEQ ID SEQ ID SEQ ID(TIM-3) E: 22-202, phosphatidyl- NO: 17 NO: 269 NO: 44 T: 203-223,serine, (22-301) C: 224-301 Galectin-9, HMGB1 CEACAM1 P13688.2 35-142IgV, S: 1-34, TIM-3 SEQ ID SEQ ID SEQ ID 145-232 IgC2, E: 35-428, NO: 18NO: 270 NO: 45 237-317 IgC2, T: 429-452, (35-526) 323-413 IgC C: 453-526TIGIT Q495A1.1 22-124 IgV S: 1-21, CD155, CD112 SEQ ID SEQ ID SEQ ID E:22-141, NO: 19 NO: 271 NO: 46 T: 142-162, (22-244) C: 163-244 PVRP15151.2 24-139 IgV, S: 1-20, TIGIT, CD226, SEQ ID SEQ ID SEQ ID (CD155)145-237 IgC2, E: 21-343, CD96, NO: 20 NO: 272 NO: 47 244-328 IgC2 T:344-367, poliovirus (21-417) C: 368-417 PVRL2 Q92692.1 32-156 IgV, S:1-31, TIGIT, CD226, SEQ ID SEQ ID SEQ ID (CD112) 162-256 IgC2, E:32-360, CD112R NO: 21 NO: 273 NO: 48 261-345 IgC2 T: 361-381, (32-538)C: 382-538 CD226 Q15762.2 19-126 IgC2, S: 1-18, CD155, CD112 SEQ ID SEQID SEQ ID 135-239 IgC2 E: 19-254, NO: 22 NO: 274 NO: 49 T: 255-275,(19-336) C: 276-336 CD2 P06729.2 25-128 IgV, S: 1-24, CD58 SEQ ID SEQ IDSEQ ID 129-209 IgC2 E: 25-209, NO: 23 NO: 275 NO: 50 T: 210-235,(25-351) C: 236-351 CD160 O95971.1 27-122 IgV S: 1-26 HVEM, MHC SEQ IDSEQ ID SEQ ID E: 27-122 family of NO: 24 NO: 276 NO: 51 proteins(27-159) CD200 P41217.4 31-141 IgV, S: 1-30, CD200R SEQ ID SEQ ID SEQ ID142-232 IgC2 E: 31-232, NO: 25 NO: 277 NO: 52 T: 233-259, (31-278) C:260-278 CD200R1 Q8TD46.2 53-139 IgV, S: 1-28, CD200 SEQ ID SEQ ID SEQ ID(CD200R) 140-228 IgC2 E: 29-243, NO: 26 NO: 278 NO: 53 T: 244-264,(29-325) C: 265-325 NCR3 O14931.1 19-126 IgC- S: 1-18, B7-H6 SEQ ID SEQID SEQ ID (NKp30) like E: 19-135, NO:27 NO: 279 NO: 54 T: 136-156,(19-201) C: 157-201 VSIG8 Q5VU13 22-141 IgV 1 S: 1-21 VISTA SEQ ID SEQID SEQ ID 146-257 E: 22-263 NO: 341 NO: 342 NO: 343 IgV 2 T: 264-284(22-414) C: 285-414

In some embodiments, the provided immunomodulatory proteins, in additionto containing a variant ICOSL polypeptide, also contains at least 2, 3,4, 5 or 6 additional immunoglobulin superfamily (IgSF) domains, such asan IgD domain of an IgSF family member set forth in Table 2. In someembodiments, the provided immunomodulatory proteins contain at least oneadditional IgSF domain (e.g. a second IgSF domain) in which at least oneadditional or second IgSF domain is an IgSF domain set forth in awild-type or unmodified IgSF domain or a specific binding fragmentthereof contained in the sequence of amino acids set forth in any of SEQID NOS: 1-27 and 341. In some embodiments, the wild-type or unmodifiedIgSF domain is an IgV domain or an IgC domain, such as an IgC1 or IgC2domain.

In some embodiments, the provided immunomodulatory proteins, in additionto containing a variant ICOSL polypeptide, also contains at least oneadditional IgSF domain (e.g. a second IgSF domain) that is a vIgD thatcontains one or more amino acid modifications (e.g. substitution,deletion or mutation) compared to an IgSF domain in a wild-type orunmodified IgSF domain, such as an IgSF domain in an IgSF family memberset forth in Table 2. In some embodiments, the additional or secondaffinity-modified IgSF domain comprises at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to a wild-type or unmodified IgSF domain or a specific bindingfragment thereof contained in the sequence of amino acids set forth inany of SEQ ID NOS: 1-27 and 341. In some embodiments, the wild-type orunmodified IgSF domain is an IgV domain or an IgC domain, such as anIgC1 or IgC2 domain. In some embodiments, the additional or second IgSFdomain is an affinity-modified IgV domain or IgC domain.

In some embodiments, the provided immunomodulatory protein contains atleast one additional or second IgSF domain that is a vIgD that containsone or more amino acid substitutions compared to an IgSF domain (e.g.IgV) of a wild-type or unmodified IgSF domain other than ICOSL.

In some embodiments, the additional or second IgSF domain contains oneor more amino acid substitutions compared to an IgSF domain in awild-type or unmodified IgSF domain, such as an IgSF domain in an IgSFfamily member set forth in Table 2. In some embodiments, the additionalor second affinity-modified IgSF domain comprises at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to a wild-type or unmodified IgSF domain or a specificbinding fragment thereof contained in the sequence of amino acids setforth in any of SEQ ID NOS: 1-27. In some embodiments, the wild-type orunmodified IgSF domain is an IgV domain or an IgC domain, such as anIgC1 or IgC2 domain. In some embodiments, the additional or second IgSFdomain is an affinity-modified IgV domain or IgC domain. Tables 3-5provide exemplary polypeptides containing one or more affinity-modifiedIgSF domains that can be used in stack constructs provided herein.

In some embodiments, the one or more additional IgSF domain (e.g. secondIgSF) domain is an IgSF domain (e.g. IgV) of another IgSF family memberthat binds or recognizes a tumor antigen. In such embodiments, the IgSFfamily member serves as a tumor-localizing moiety, thereby bringing thevIgD of ICOSL in close proximity to immune cells in the tumormicroenvironment. In some embodiments, the additional IgSF domain (e.g.second IgSF) domain is an IgSF domain of NkP30, which binds orrecognizes B7-H6 expressed on a tumor cell. In some embodiments, the atleast one additional (e.g. second) IgSF domain, e.g. NkP30, is a vIgDthat contains one or more amino acid modifications (e.g. substitutions,deletions or additions). In some embodiments, the one or more amino acidmodifications increase binding affinity and/or selectivity to B7-H6compared to unmodified IgSF domain, e.g. NkP30, such as by at least orat least about 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold 40-fold or50-fold.

TABLE 3 Exemplary variant CD80 polypeptides IgV ECD SEQ SEQ IDMutation(s) ID NO NO Wild-type 28 152 L70Q/A91G 55 153 L70Q/A91G/T130A56 L70Q/A91G/I118A/T120S/T130A 57 V4M/L70Q/A91G/T120S/T130A 58 154L70Q/A91G/T120S/T130A 59 V20L/L70Q/A91S/T120S/T130A 60 155S44P/L70Q/A91G/T130A 61 156 L70Q/A91G/E117G/T120S/T130A 62A91G/T120S/T130A 63 157 L70R/A91G/T120S/T130A 64 158L70Q/E81A/A91G/T120S/I127T/T130A 65 159 L70Q/Y87N/A91G/T130A 66 160T28S/L70Q/A91G/E95K/T120S/T130A 67 161 N63S/L70Q/A91G/T120S/T130A 68 162K36E/I67T/L70Q/A91G/T120S/T130A/N152T 69 163 E52G/L70Q/A91G/T120S/T130A70 164 K37E/F59S/L70Q/A91G/T120S/T130A 71 165 A91G/S103P 72 K89E/T130A73 166 A91G 74 D60V/A91G/T120S/T130A 75 167 K54M/A91G/T120S 76 168M38T/L70Q/E77G/A91G/T120S/T130A/N152T 77 169R29H/E52G/L70R/E88G/A91G/T130A 78 170 Y31H/T41G/L70Q/A91G/T120S/T130A 79171 V68A/T110A 80 172 S66H/D90G/T110A/F116L 81 173 R29H/E52G/T120S/T130A82 174 A91G/L102S 83 I67T/L70Q/A91G/T120S 84 175L70Q/A91G/T110A/T120S/T130A 85M38V/T41D/M43I/W50G/D76G/V83A/K89E/T120S/T130A 86 176 V22A/L70Q/S121P 87177 A12V/S15F/Y31H/T41G/T130A/P137L/N152T 88 178I67F/L70R/E88G/A91G/T120S/T130A 89 179 E24G/L25P/L70Q/T120S 90 180A91G/F92L/F108L/T120S 91 181R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/ 92 182F92P/K93V/R94L/I118T/N149SR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/ 93F92P/K93V/R94L/N144S/N149SR29D/Y31L/Q33H/K36G/M38I/T41A/M42T/M43R/M47T/E81V/L85R/K89N/ 94 183A91T/F92P/K93V/R94L/L148S/N149SE24G/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/F59L/E81V/L85R/ 95 184K89N/A91T/F92P/K93V/R94L/H96R/N149S/C182SR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/ 96F92P/K93V/R94L/N149S R29V/M43Q/E81R/L85I/K89R/D90L/A91E/F92N/K93Q/R94G97 185 T41I/A91G 98 186 K89R/D90K/A91G/F92Y/K93R/N122S/N177S 99 187K89R/D90K/A91G/F92Y/K93R 100K36G/K37Q/M38I/F59L/E81V/L85R/K89N/A91T/F92P/K93V/R94L/E99G/ 101 188T130A/N149S E88D/K89R/D90K/A91G/F92Y/K93R 102 189, 543K36G/K37Q/M38I/L40M 103 190 K36G 104 191R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S 105 192A12T/H18L/M43V/F59L/E77K/P109S/I118T 106 193R29V/Y31F/K36G/M38L/M43Q/E81R/V83I/L85I/K89R/D90L/A91E/F92N/ 107 194K93Q/R94G V68M/L70P/L72P/K86E 108 195 L70Q/A91G/N144D 508L70Q/A91G/I118A/T120S/T130A/K169E 509V4M/L70Q/A91G/I118V/T120S/T130A/K169E 510L70Q/A91G/I118V/T120S/T130A/K169E 511 L70Q/A91G/I118V/T120S/T130A 512V20L/L70Q/A91S/I118V/T120S/T130A 513 L70Q/A91G/E117G/I118V/T120S/T130A514 A91G/I118V/T120S/T130A 515 L70R/A91G/I118V/T120S/T130A/T199S 516L70Q/E81A/A91G/I118V/T120S/I127T/T130A 517T28S/L70Q/A91G/E95K/I118V/T120S/I126V/T130A/K169E 518N63S/L70Q/A91G/S114T/I118V/T120S/T130A 519K36E/I67T/L70Q/A91G/I118V/T120S/T130A/N152T 520E52G/L70Q/A91G/D107N/I118V/T120S/T130A/K169E 521K37E/F59S/L70Q/A91G/I118V/T120S/T130A/K185E 522D60V/A91G/I118V/T120S/T130AK169E 523 K54M/L70Q/A91G/Y164H/T120S 524M38T/L70Q/E77G/A91G/I118V/T120S/T130A/N152T 525Y31H/T41G/M43L/L70Q/A91G/I118V/T120S/I126V/T130A 526LS656H/D90G/T110A/F116L 527 R29H/E52G/D90N/I118V/T120S/T130A 528R29H/E52G/D90N/I118V/T120S/T130A 529 I67T/L70Q/A91G/I118V/T120S 530L70Q/A91G/T110A/I118V/T120S/T130A 531M38V/T41D/M43I/W50G/D76G/V83A/K89E/I118V/T120S/I126V/T130A 532A12V/S15F/Y31H/M38L/T41G/M43L/D90N/T130A/P137L/N149D/N152T 533I67F/L70R/E88G/A91G/I118V/T120S/T130A 534E24G/L25P/L70Q/A91G/I118VT120S/N152T 535 A91G/F92L/F108L/I118V/T120S 536E88D/K89R/D90K/A91G/F92Y/K93R/N122S/N177S 537K36G/K37Q/M38I/L40M/F59L/E81V/L85R/K89N/A91T/F92P/K93V/R94L/ 539E99G/T130A/N149S K36G/L40M 540 542, 544

TABLE 4 Exemplary variant NKp30 polypeptides IgC-like ECD domain SEQ IDSEQ ID Mutation(s) NO NO Wild-type 54 214 L30V/A60V/S64P/S86G 143 215L30V 144 216 A60V 145 217 S64P 146 218 S86G 147 219

TABLE 5 Exemplary variant CD86 polypeptides ECD IgV SEQ ID SEQ IDMutation(s) NO NO Wild-type 29 220 Q35H/H90L/Q102H 148 221 Q35H 149 222H90L 150 223 Q102H 151 224

The number of such non-affinity modified or affinity modified IgSFdomains present in a “stacked” immunomodulatory protein construct(whether non-wild type combinations or non-wild type arrangements) is atleast 2, 3, 4, or 5 and in some embodiments exactly 2, 3, 4, or 5 IgSFdomains (whereby determination of the number of affinity modified IgSFdomains disregards any non-specific binding fractional sequences thereofand/or substantially immunologically inactive fractional sequencesthereof).

In some embodiments of a stacked immunomodulatory protein providedherein, the number of IgSF domains is at least 2 wherein the number ofaffinity modified and the number of non-affinity modified IgSF domainsis each independently at least: 0, 1, 2, 3, 4, 5, or 6. Thus, the numberof affinity modified IgSF domains and the number of non-affinitymodified IgSF domains, respectively, (affinity modified IgSF domain:non-affinity modified IgSF domain), can be exactly or at least: 2:0(affinity modified: wild-type), 0:2, 2:1, 1:2, 2:2, 2:3, 3:2, 2:4, 4:2,1:1, 1:3, 3:1, 1:4, 4:1, 1:5, or 5:1.

In some embodiments of a stacked immunomodulatory protein, at least twoof the non-affinity modified and/or affinity modified IgSF domains areidentical IgSF domains.

In some embodiments, a stacked immunomodulatory protein provided hereincomprises at least two affinity modified and/or non-affinity modifiedIgSF domains from a single IgSF member but in a non-wild-typearrangement (alternatively, “permutation”). One illustrative example ofa non-wild type arrangement or permutation is an immunomodulatoryprotein comprising a non-wild-type order of affinity modified and/ornon-affinity modified IgSF domain sequences relative to those found inthe wild-type ICOSL whose IgSF domain sequences served as the source ofthe variant IgSF domains as provided herein. Thus, in one example, theimmunomodulatory protein can comprise an IgV proximal and an IgC distalto the transmembrane domain albeit in a non-affinity modified and/oraffinity modified form. The presence, in an immunomodulatory proteinprovided herein, of both non-wild-type combinations and non-wild-typearrangements of non-affinity modified and/or affinity modified IgSFdomains is also within the scope of the provided subject matter.

In some embodiments of a stacked immunomodulatory protein, thenon-affinity modified and/or affinity modified IgSF domains arenon-identical (i.e., different) IgSF domains. Non-identical affinitymodified IgSF domains specifically bind, under specific bindingconditions, different cognate binding partners and are “non-identical”irrespective of whether or not the wild-type or unmodified IgSF domainsfrom which they are engineered was the same. Thus, for example, anon-wild-type combination of at least two non-identical IgSF domains inan immunomodulatory protein can comprise at least one IgSF domainsequence whose origin is from and unique to one ICOSL, and at least oneof a second IgSF domain sequence whose origin is from and unique toanother IgSF family member that is not ICOSL, wherein the IgSF domainsof the immunomodulatory protein are in non-affinity modified and/oraffinity modified form. However, in alternative embodiments, the twonon-identical IgSF domains originate from the same IgSF domain sequencebut at least one is affinity modified such that they specifically bindto different cognate binding partners.

A plurality of non-affinity modified and/or affinity modified IgSFdomains in a stacked immunomodulatory protein polypeptide chain need notbe covalently linked directly to one another. In some embodiments, anintervening span of one or more amino acid residues indirectlycovalently bonds the non-affinity modified and/or affinity modified IgSFdomains to each other. The linkage can be via the N-terminal toC-terminal residues.

In some embodiments, the linkage can be made via side chains of aminoacid residues that are not located at the N-terminus or C-terminus ofthe non-affinity modified and/or affinity modified IgSF domain. Thus,linkages can be made via terminal or internal amino acid residues orcombinations thereof.

In some embodiments, the two or more IgSF domain, including a vIgD ofICOSL and one or more additional IgSF domain (e.g. second variant IgSFdomain) from another IgSF family member, are covalently ornon-covalently linked. In some embodiments, the two or more IgSF domainsare linked directly or indirectly, such as via a linker. In someembodiments, an intervening span of one or more amino acid residuesindirectly covalently bonds IgSF domains to each other. The linkage canbe via the N-terminal to C-terminal residues. In some embodiments, thelinkage can be made via side chains of amino acid residues that are notlocated at the N-terminus or C-terminus of the IgSF domain(s). Thus,linkages can be made via terminal or internal amino acid residues orcombinations thereof.

In some embodiments, one or more “peptide linkers” link the vIgD ofICOSL and an additional IgSF domain (e.g. second variant IgSF domain).In some embodiments, a peptide linker can be a single amino acid residueor greater in length. In some embodiments, the peptide linker has atleast one amino acid residue but is no more than 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues inlength. In some embodiments, the linker is (in one-letter amino acidcode): GGGGS (“4GS”) or multimers of the 4GS linker, such as repeats of2, 3, 4, or 5 4GS linkers. In some embodiments, the peptide linker is(GGGGS)₂ or (GGGGS)₃. In some embodiments, the linker also can include aseries of alanine residues alone or in addition to another peptidelinker (such as a 4GS linker or multimer thereof). In some embodiments,the number of alanine residues in each series is: 2, 3, 4, 5, or 6alanines.

In some embodiments, the non-affinity modified and/or affinity modifiedIgSF domains are linked by “wild-type peptide linkers” inserted at theN-terminus and/or C-terminus of the first and/or second non-affinitymodified and/or affinity modified IgSF domains. In some embodiments,there is present a leading peptide linker inserted at the N-terminus ofthe first IgSF domain and/or a first trailing sequence inserted at theC-terminus of the first non-affinity modified and/or affinity modifiedIgSF domain. In some embodiments, there is present a second leadingpeptide linker inserted at the N-terminus of the second IgSF domainand/or a second trailing sequence inserted at the C-terminus of thesecond non-affinity modified and/or affinity modified IgSF domain. Whenthe first and second non-affinity modified and/or affinity modified IgSFdomains are derived from the same parental protein and are connected inthe same orientation, wild-type peptide linkers between the first andsecond non-affinity modified and/or affinity modified IgSF domains arenot duplicated. For example, when the first trailing wild-type peptidelinker and the second leading wild-type peptide linker are the same, theType II immunomodulatory protein does not comprise either the firsttrailing wild-type peptide linker or the second leading wild-typepeptide linker.

In some embodiments, the Type II immunomodulatory protein comprises afirst leading wild-type peptide linker inserted at the N-terminus of thefirst non-affinity modified and/or affinity modified IgSF domain,wherein the first leading wild-type peptide linker comprises at least 5(such as at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ormore) consecutive amino acids from the intervening sequence in thewild-type protein from which the first non-affinity modified and/oraffinity modified IgSF domain is derived between the parental IgSFdomain and the immediately preceding domain (such as a signal peptide oran IgSF domain). In some embodiments, the first leading wild-typepeptide linker comprises the entire intervening sequence in thewild-type protein from which the first non-affinity modified and/oraffinity modified IgSF domain is derived between the parental IgSFdomain and the immediately preceding domain (such as a signal peptide oran IgSF domain).

In some embodiments, the Type II immunomodulatory protein furthercomprises a first trailing wild-type peptide linker inserted at theC-terminus of the first non-affinity modified and/or affinity modifiedIgSF domain, wherein the first trailing wild-type peptide linkercomprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more) consecutive amino acids from the interveningsequence in the wild-type protein from which the first non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately following domain (such as anIgSF domain or a transmembrane domain). In some embodiments, the firsttrailing wild-type peptide linker comprises the entire interveningsequence in the wild-type protein from which the first non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately following domain (such as anIgSF domain or a transmembrane domain).

In some embodiments, the Type II immunomodulatory protein furthercomprises a second leading wild-type peptide linker inserted at theN-terminus of the second non-affinity modified and/or affinity modifiedIgSF domain, wherein the second leading wild-type peptide linkercomprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more) consecutive amino acids from the interveningsequence in the wild-type protein from which the second non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately preceding domain (such as asignal peptide or an IgSF domain). In some embodiments, the secondleading wild-type peptide linker comprises the entire interveningsequence in the wild-type protein from which the second non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately preceding domain (such as asignal peptide or an IgSF domain).

In some embodiments, the Type II immunomodulatory protein furthercomprises a second trailing wild-type peptide linker inserted at theC-terminus of the second non-affinity modified and/or affinity modifiedIgSF domain, wherein the second trailing wild-type peptide linkercomprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more) consecutive amino acids from the interveningsequence in the wild-type protein from which the second non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately following domain (such as anIgSF domain or a transmembrane domain). In some embodiments, the secondtrailing wild-type peptide linker comprises the entire interveningsequence in the wild-type protein from which the second non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately following domain (such as anIgSF domain or a transmembrane domain).

Exemplary of a leading sequence and trailing sequence for a Type IIprotein containing a CD80 IgSF domain is set forth in SEQ ID NO:231 andSEQ ID NO:232. Exemplary of a leading sequence and trailing sequence fora Type II protein containing an ICOSL IgSF domain is set forth in SEQ IDNO: 233 and 234. Exemplary of a leading sequence and a trailing sequencefor a Type II protein containing an CD86 IgSF domain is set forth in anyof SEQ ID NOS: 236-238. Exemplary of a wild-type linker sequence for aType II protein containing an NKp30 IgSF domain is set forth in SEQ IDNO:235.

In some embodiments, the two or more IgSF domain, including a vIgD ofICOSL and one or more additional IgSF domain (e.g. second variant IgSFdomain) from another IgSF family member, are linked or attached to an Fcto form a dimeric multi-domain stack immunomodulatory protein. In someembodiments, the variant ICOSL polypeptide and second IgSF domain areindependently linked, directly or indirectly, to the N- or C-terminus ofan Fc subunit. In some embodiments, the variant ICOSL polypeptide andsecond IgSF domain are linked, directly or indirectly, and one of thevariant ICOSL or second IgSF domain is also linked, directly orindirectly, to the N- or C-terminus of an Fc subunit. In someembodiments, linkage to the Fc is via a peptide linker, e.g. a peptidelinker, such as described above. In some embodiments, linkage betweenthe variant ICOSL and second IgSF domain is via a peptide linker, e.g. apeptide linker, such as described above. In some embodiments, the vIgDof ICOSL, the one or more additional IgSF domains, and the Fc domain canbe linked together in any of numerous configurations as depicted in FIG.16. Exemplary configurations are described in the Examples.

In some embodiments, the stacked immunomodulatory protein is a dimerformed by two stacked immunomodulatory Fc fusion polypeptides. Alsoprovided are nucleic acid molecules encoding any of the stackedimmunomodulatory proteins. In some embodiments, the dimeric multi-domainstack immunomodulatory protein can be produced in cells by expression,or in some cases co-expression, of stack immunomodulatory Fc fusionpolypeptides, such as described above in according with generatingdimeric Fc fusion proteins.

In some embodiments, the dimeric multi-domain stack immunomodulatoryprotein is divalent for each Fc subunit, monovalent for each subunit, ordivalent for one subunit and tetravalent for the other.

In some embodiments, the dimeric multi-domain stack immunomodulatoryprotein is a homodimeric multi-domain stack Fc protein. In someembodiments, the dimeric multi-domain stack immunomodulatory proteincomprises a first stack immunomodulatory Fc fusion polypeptide and asecond stack immunomodulatory Fc fusion polypeptide in which the firstand second polypeptide are the same. In some embodiments, the Fc portionof the polypeptide can be any Fc as described above.

In some embodiments, the multi-domain stack molecule is heterodimeric,comprising two different Fc polypeptides wherein at least one is an Fcpolypeptide containing at least one variant ICOSL polypeptide and/or atleast one second IgSF domain (e.g. second variant IgSF domain). In someembodiments, the multi-domain stack molecule contains a first Fcpolypeptide containing a variant ICOSL and a second IgSF domain and asecond Fc polypeptide containing the variant ICOSL and the second IgSFdomain. In some embodiments, the multi-domain stack molecule contains afirst Fc polypeptide containing a variant ICOSL polypeptide and a secondIgSF domain and a second Fc polypeptide that is not linked to either avariant ICOSL polypeptide or second IgSF domain.

In some embodiments, the multi-domain stack molecule contains a first Fcpolypeptide containing 1, 2, 3, 4 or more variant ICOSL polypeptidesand/or 1, 2, 3, 4 or more second IgSF domains, wherein the total numberof IgSF domains in the first stack Fc polypeptide is greater than 2, 3,4, 5, 6 or more. In one example of such an embodiment, the second stackFc polypeptide contains 1, 2, 3, 4 or more variant ICOSL polypeptidesand/or 1, 2, 3, 4 or more second IgSF domains, wherein the total numberof IgSF domains in the second stack Fc polypeptide is greater than 2, 3,4, 5, 6 or more. In another example of such an embodiment, the second Fcpolypeptide is not linked to either a variant ICOSL polypeptide orsecond IgSF domain.

In some embodiments, the heterodimeric stack molecule contains a firststack immunomodulatory Fc fusion polypeptide and a second stackimmunomodulatory Fc fusion polypeptide in which the first and secondpolypeptide are different. In some embodiments, a heterodimeric stackmolecule contains a first Fc subunit containing a first variant ICOSLpolypeptide and/or second IgSF domain (e.g. second variant IgSF domain)and a second Fc subunit containing the other of the first variant ICOSLpolypeptide or the second IgSF domain. In some embodiments, theheterodimeric stack molecule contains a first stack immunomodulatory Fcfusion polypeptide and a second stack immunomodulatory Fc fusionpolypeptide in which the first and second polypeptide are different. Insome embodiments, a heterodimeric stack molecule contains a first Fcsubunit containing a first variant ICOSL polypeptide and/or second IgSFdomain (e.g. second variant IgSF domain) and a second Fc subunitcontaining both the first variant ICOSL polypeptide and second IgSFdomain (e.g. second variant IgSF domain) but in a different orientationor configuration from the first Fc subunit.

In some embodiments, the Fc domain of one or both of the first andsecond stacked immunomodulatory Fc fusion polypeptide comprises amodification (e.g. substitution) such that the interface of the Fcmolecule is modified to facilitate and/or promote heterodimerization. Insome embodiments, modifications include introduction of a protuberance(knob) into a first Fc polypeptide and a cavity (hole) into a second Fcpolypeptide such that the protuberance is positionable in the cavity topromote complexing of the first and second Fc-containing polypeptides.Amino acids targeted for replacement and/or modification to createprotuberances or cavities in a polypeptide are typically interface aminoacids that interact or contact with one or more amino acids in theinterface of a second polypeptide.

In some embodiments, a first polypeptide that is modified to containprotuberance (hole) amino acids include replacement of a native ororiginal amino acid with an amino acid that has at least one side chainwhich projects from the interface of the first polypeptide and istherefore positionable in a compensatory cavity (hole) in an adjacentinterface of a second polypeptide. Most often, the replacement aminoacid is one which has a larger side chain volume than the original aminoacid residue. One of skill in the art knows how to determine and/orassess the properties of amino acid residues to identify those that areideal replacement amino acids to create a protuberance. In someembodiments, the replacement residues for the formation of aprotuberance are naturally occurring amino acid residues and include,for example, arginine (R), phenylalanine (F), tyrosine (Y), ortyrptophan (W). In some examples, the original residue identified forreplacement is an amino acid residue that has a small side chain suchas, for example, alanine, asparagines, aspartic acid, glycine, serine,threonine, or valine.

In some embodiments, a second polypeptide that is modified to contain acavity (hole) is one that includes replacement of a native or originalamino acid with an amino acid that has at least one side chain that isrecessed from the interface of the second polypeptide and thus is ableto accommodate a corresponding protuberance from the interface of afirst polypeptide. Most often, the replacement amino acid is one whichhas a smaller side chain volume than the original amino acid residue.One of skill in the art knows how to determine and/or assess theproperties of amino acid residues to identify those that are idealreplacement residues for the formation of a cavity. Generally, thereplacement residues for the formation of a cavity are naturallyoccurring amino acids and include, for example, alanine (A), serine (S),threonine (T) and valine (V). In some examples, the original amino acididentified for replacement is an amino acid that has a large side chainsuch as, for example, tyrosine, arginine, phenylalanine, or typtophan.

The CH3 interface of human IgG1, for example, involves sixteen residueson each domain located on four anti-parallel (3-strands which buries1090 A2 from each surface (see e.g., Deisenhofer et al. (1981)Biochemistry, 20:2361-2370; Miller et al., (1990) J Mol. Biol., 216,965-973; Ridgway et al., (1996) Prot. Engin., 9: 617-621; U.S. Pat. No.5,731,168). Modifications of a CH3 domain to create protuberances orcavities are described, for example, in U.S. Pat. No. 5,731,168;International Patent Applications WO98/50431 and WO 2005/063816; andRidgway et al., (1996) Prot. Engin., 9: 617-621. In some examples,modifications of a CH3 domain to create protuberances or cavities aretypically targeted to residues located on the two central anti-parallelβ-strands. The aim is to minimize the risk that the protuberances whichare created can be accommodated by protruding into the surroundingsolvent rather than being accommodated by a compensatory cavity in thepartner CH3 domain.

In some embodiments, the heterodimeric molecule contains a T366Wmutation in the CH3 domain of the “knobs chain” and T366S, L368A, Y407Vmutations in the CH3 domain of the “hole chain”. In some cases, anadditional interchain disulfide bridge between the CH3 domains can alsobe used (Merchant, A. M., et al., Nature Biotech. 16 (1998) 677-681)e.g. by introducing a Y349C mutation into the CH3 domain of the “knobs”or “hole” chain and a E356C mutation or a S354C mutation into the CH3domain of the other chain. In some embodiments, the heterodimericmolecule contains S354C, T366W mutations in one of the two CH3 domainsand Y349C, T366S, L368A, Y407V mutations in the other of the two CH3domains. In some embodiments, the heterodimeric molecule comprisesE356C, T366W mutations in one of the two CH3 domains and Y349C, T366S,L368A, Y407V mutations in the other of the two CH3 domains. In someembodiments, the heterodimeric molecule comprises Y349C, T366W mutationsin one of the two CH3 domains and E356C, T366S, L368A, Y407V mutationsin the other of the two CH3 domains. In some embodiments, theheterodimeric molecule comprises Y349C, T366W mutations in one of thetwo CH3 domains and S354C, T366S, L368A, Y407V mutations in the other ofthe two CH3 domains. Examples of other knobs-in-holes technologies areknown in the art, e.g. as described by EP 1 870 459 A1.

In some embodiments, the Fc subunits of the heterodimeric moleculeadditionally can contain one or more other Fc mutation, such as anydescribed above. In some embodiments, the heterodimer molecule containsan Fc subunit with a mutation that reduces effector function.

In some embodiments, an Fc variant containing CH3 protuberance/cavitymodifications can be joined to a stacked immunomodulatory polypeptideanywhere, but typically via its N- or C-terminus, to the N- orC-terminus of a first and/or second stacked immunomodulatorypolypeptide, such as to form a fusion polypeptide. The linkage can bedirect or indirect via a linker. Typically, a knob and hole molecule isgenerated by co-expression of a first stacked immunomodulatorypolypeptide linked to an Fc variant containing CH3 protuberancemodification(s) with a second stacked immunomodulatory polypeptidelinked to an Fc variant containing CH3 cavitity modification(s).

C. Conjugates and Fusions of Variant Polypeptides and ImmunomodulatoryProteins

In some embodiments, the variant polypeptides provided herein, which areimmunomodulatory proteins comprising variants of an Ig domain of theIgSF family (vIgD), can be conjugated with or fused with a moiety, suchas an effector moiety, such as another protein, directly or indirectly,to form a conjugate (“IgSF conjugate”). In some embodiments, theattachment can be covalent or non-covalent, e.g., via abiotin-streptavidin non-covalent interaction. In some embodiments of aICOSL-Fc variant fusion, any one or combination of any two or more ofthe foregoing conjugates can be attached to the Fc or to the variantCD80 polypeptide or to both

In some embodiments, the moiety can be a targeting moiety, a smallmolecule drug (non-polypeptide drug of less than 500 daltons molarmass), a toxin, a cytostatic agent, a cytotoxic agent, animmunosuppressive agent, a radioactive agent suitable for diagnosticpurposes, a radioactive metal ion for therapeutic purposes, aprodrug-activating enzyme, an agent that increases biological half-life,or a diagnostic or detectable agent.

In some embodiments the effector moiety is a therapeutic agent, such asa cancer therapeutic agent, which is either cytotoxic, cytostatic orotherwise provides some therapeutic benefit. In some embodiments, theeffector moiety is a targeting moiety or agent, such as an agent thattargets a cell surface antigen, e.g., an antigen on the surface of atumor cell. In some embodiments, the effector moiety is a label, whichcan generate a detectable signal, either directly or indirectly. In someembodiments, the effector moiety is a toxin. In some embodiments, theeffector moiety is a protein, peptide, nucleic acid, small molecule ornanoparticle.

In some embodiments, 1, 2, 3, 4, 5 or more effector moieties, which canbe the same or different, are conjugated, linked or fused to the variantpolypeptide or protein to form an IgSF conjugate. In some embodiments,such effector moieties can be attached to the variant polypeptide orimmunomodulatory protein using various molecular biological or chemicalconjugation and linkage methods known in the art and described below. Insome embodiments, linkers such as peptide linkers, cleavable linkers,non-cleavable linkers or linkers that aid in the conjugation reaction,can be used to link or conjugate the effector moieties to the variantpolypeptide or immunomodulatory protein.

In some embodiments, the IgSF conjugate comprises the followingcomponents: (protein or polypeptide), (L)_(q) and (effector moiety)_(m),wherein the protein or polypeptide is any of the described variantpolypeptides or immunomodulatory proteins capable of binding one or morecognate counter structure ligands as described; L is a linker forlinking the protein or polypeptide to the moiety; m is at least 1; q is0 or more; and the resulting IgSF conjugate binds to the one or morecounter structure ligands. In particular embodiments, m is 1 to 4 and qis 0 to 8.

In some embodiments, there is provided an IgSF conjugate comprising avariant polypeptide or immunomodulatory protein provided hereinconjugated with a targeting agent that binds to a cell surface molecule,for example, for targeted delivery of the variant polypeptide orimmunomodulatory protein to a specific cell. In some embodiments, thetargeting agent is a molecule(s) that has the ability to localize andbind to a molecule present on a normal cell/tissue and/or tumorcell/tumor in a subject. In other words, IgSF conjugates comprising atargeting agent can bind to a ligand (directly or indirectly), which ispresent on a cell, such as a tumor cell. The targeting agents of theinvention contemplated for use include antibodies, polypeptides,peptides, aptamers, other ligands, or any combination thereof, that canbind a component of a target cell or molecule.

In some embodiments, the targeting agent binds a tumor cell(s) or canbind in the vicinity of a tumor cell(s) (e.g., tumor vasculature ortumor microenvironment) following administration to the subject. Thetargeting agent may bind to a receptor or ligand on the surface of thecancer cell. In another aspect of the invention, a targeting agent isselected which is specific for a noncancerous cells or tissue. Forexample, a targeting agent can be specific for a molecule presentnormally on a particular cell or tissue. Furthermore, in someembodiments, the same molecule can be present on normal and cancercells. Various cellular components and molecules are known. For example,if a targeting agent is specific for EGFR, the resulting IgSF conjugatecan target cancer cells expressing EGFR as well as normal skin epidermalcells expressing EGFR. Therefore, in some embodiments, an IgSF conjugateof the invention can operate by two separate mechanisms (targetingcancer and non-cancer cells).

In various aspects of the invention disclosed herein an IgSF conjugateof the invention comprises a targeting agent which can bind/target acellular component, such as a tumor antigen, a bacterial antigen, aviral antigen, a mycoplasm antigen, a fungal antigen, a prion antigen,an antigen from a parasite. In some aspects, a cellular component,antigen or molecule can each be used to mean, a desired target for atargeting agent. For example, in various embodiments, a targeting agentis specific for or binds to a component, which includes but is notlimited to, epidermal growth factor receptor (EGFR, ErbB-1, HER1),ErbB-2 (HER2/neu), ErbB-3/HER3, ErbB-4/HER4, EGFR ligand family;insulin-like growth factor receptor (IGFR) family, IGF-binding proteins(IGFBPs), IGFR ligand family; platelet derived growth factor receptor(PDGFR) family, PDGFR ligand family; fibroblast growth factor receptor(FGFR) family, FGFR ligand family, vascular endothelial growth factorreceptor (VEGFR) family, VEGF family; HGF receptor family; TRK receptorfamily; ephrin (EPH) receptor family; AXL receptor family; leukocytetyrosine kinase (LTK) receptor family; TIE receptor family, angiopoietin1,2; receptor tyrosine kinase-like orphan receptor (ROR) receptorfamily, e.g. ROR1; CD171 (L1CAM); B7-H6 (NCR3LG1); PD-L1, tumorglycosylation antigen, e.g. sTn or Tn, such as sTn Ag of MUCl; LHR(LHCGR); phosphatidylserine, discoidin domain receptor (DDR) family; RETreceptor family; KLG receptor family; RYK receptor family; MuSK receptorfamily; Transforming growth factor-α (TGF-α) receptors, TGF-β; Cytokinereceptors, Class I (hematopoietin family) and Class II (interferon/IL-10family) receptors, tumor necrosis factor (TNF) receptor superfamily(TNFRSF), death receptor family; cancer-testis (CT) antigens,lineage-specific antigens, differentiation antigens, alpha-actinin-4,ARTC1, breakpoint cluster region-Abelson (Bcr-abl) fusion products,B-RAF, caspase-5 (CASP-5), caspase-8 (CASP-8), β-catenin (CTNNB1), celldivision cycle 27 (CDC27), cyclin-dependent kinase 4 (CDK4), CDKN2A,COA-I, dek-can fusion protein, EFTUD-2, Elongation factor 2 (ELF2), Etsvariant gene 6/acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusionprotein, fibronectin (FN), e.g. the extradomain A (EDA) of fibronectin,GPNMB, low density lipid receptor/GDP-L fucose: β-Dgalactose2-α-Lfucosyltransferase (LDLR/FUT) fusion protein, HLA-A2. arginine toisoleucine exchange at residue 170 of the α-helix of the a2-domain inthe HLA-A2gene (HLA-A*201-R170I), HLA-A1 1, heat shock protein 70-2mutated (HSP70-2M), K1AA0205, MART2, melanoma ubiquitous mutated 1, 2, 3(MUM-I, 2, 3), prostatic acid phosphatase (PAP), neo-PAP, Myosin classI, NFYC, OGT, OS-9, pml-RARalpha fusion protein, PRDX5, PTPRK, K-ras(KRAS2), N-ras (NRAS), HRAS, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2fusion protein, Triosephosphate Isomerase, BAGE, BAGK-1, BAGE-2,3,4,5,GAGE-1,2,3,4,5,6,7,8, GnT-V (aberrant N-acetyl glucosaminyl transferaseV, MGAT5), HERV-K-MEL, KK-LC, KM-HN-I, LAGE, LAGE-I, CTL-recognizedantigen on melanoma (CAMEL), MAGE-A1 (MAGE-I), MAGE-A2, MAGE-A3,MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11,MAGE-A12, MAGE-3, MAGE-B1, MAGE-B2, MAGE-B5, MAGE-B6, MAGE- C1, MAGE-C2,mucin 1 (MUC1), MART-1/Melan-A (MLANA), gplOO, gplOO/Pmell7 (SILV),tyrosinase (TYR), TRP-I, HAGE, NA-88, NY-ESO-I, NY-ESO-1/LAGE-2, SAGE,Sp17, SSX-1,2,3,4, TRP2-INT2, carcino-embryonic antigen (CEA),Kallikrein 4, mammaglobin-A, OA1, prostate specific antigen (PSA),TRP-1/gp75, TRP-2, adipophilin, interferon inducible protein absent inmelanoma 2 (AIM-2), BING-4, CPSF, cyclin D1, epithelial cell adhesionmolecule (Ep-CAM), EphA3, fibroblast growth factor-5 (FGF-5),glycoprotein 250 (gp250), EGFR (ERBB1), HER-2/neu (ERBB2), interleukin13 receptor a2 chain (IL13Rα2), IL-6 receptor, intestinal carboxylesterase (iCE), alpha-feto protein (AFP), M-CSF, mdm-2, MUC1, p53(TP53), PBF, PRAME, PSMA, RAGE-I, RNF43, RU2AS, SOX1O, STEAP1, survivin(BIRC5), human telomerase reverse transcriptase (hTERT), telomerase,Wilms' tumor gene (WT1), SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1,CTAGE-I, CSAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15q14, HCA661, LDHC,MORC, SGY-I, SPOl1, TPX1, NY-SAR-35, FTHL17, NXF2, TDRD1, TEX15, FATE,TPTE, immunoglobulin idiotypes, Bence-Jones protein, estrogen receptors(ER), androgen receptors (AR), CD40, CD30, CD20, CD 19, CD33, cancerantigen 72-4 (CA 72-4), cancer antigen 15-3 (CA 15-3), cancer antigen27-29 (CA 27-29), cancer antigen 125 (CA 125), cancer antigen 19-9 (CA19-9), (3-human chorionic gonadotropin, β-2 microglobulin, squamous cellcarcinoma antigen, neuron-specific enolase, heat shock protein gp96,GM2, sargramostim, CTLA-4, 707 alanine proline (707-AP), adenocarcinomaantigen recognized by T cells 4 (ART-4), carcinoembryogenic antigenpeptide-1 (CAP-I), calcium-activated chloride channel-2 (CLCA2),cyclophilin B (Cyp-B), human signet ring tumor-2 (HST-2), Humanpapilloma virus (HPV) proteins (HPV-E6, HPV-E7, major or minor capsidantigens, others), Epstein-Barr virus (EBV) proteins (EBV latentmembrane proteins—LMP1, LMP2; others), Hepatitis B or C virus proteins,and HIV proteins.

In some embodiments, an IgSF conjugate, through its targeting agent,will bind a cellular component of a tumor cell, tumor vasculature ortumor microenvironment, thereby promoting killing of targeted cells viamodulation of the immune response, (e.g., by activation ofco-stimulatory molecules or inhibition of negative regulatory moleculesof immune cell activation), inhibition of survival signals (e.g., growthfactor or cytokine or hormone receptor antagonists), activation of deathsignals, and/or immune-mediated cytotoxicity, such as through antibodydependent cellular cytotoxicity. Such IgSF conjugates can functionthrough several mechanisms to prevent, reduce or eliminate tumor cells,such as to facilitate delivery of conjugated effector moieties to thetumor target, such as through receptor-mediated endocytosis of the IgSFconjugate; or such conjugates can recruit, bind, and/or activate immunecells (e.g. NK cells, monocytes/macrophages, dendritic cells, T cells, Bcells). Moreover, in some instances one or more of the foregoingpathways may operate upon administration of one or more IgSF conjugatesof the invention.

In some embodiments, an IgSF conjugate, through its targeting agent,will be localized to, such as bind to, a cellular component of a tumorcell, tumor vasculature or tumor microenvironment, thereby modulatingcells of the immune response in the vicinity of the tumor. In someembodiments, the targeting agent facilitates delivery of the conjugatedIgSF (e.g. vIgD) to the tumor target, such as to interact with itscognate binding partner to alter signaling of immune cells (e.g. NKcells, monocytes/macrophages, dendritic cells, T cells, B cells) bearingthe cognate binding partner. In some embodiments, localized deliveryagonizes or stimulates the costimulatory receptor.

In some embodiments, the targeting agent is an immunoglobulin. As usedherein, the term “immunoglobulin” includes natural or artificial mono-or polyvalent antibodies including, but not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, single chain Fv (scFv);anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto antibodies of the invention), and epitope-binding fragments of any ofthe above. The term “antibody,” as used herein, refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules,e.g., molecules that contain an antigen binding site thatimmunospecifically binds an antigen. The immunoglobulin molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass ofimmunoglobulin molecule.

In some embodiments, an IgSF conjugate, through its antibody targetingmoiety, will bind a cellular component of a tumor cell, tumorvasculature or tumor microenvironment, thereby promoting apoptosis oftargeted cells via modulation of the immune response, (e.g., byactivation of co-stimulatory molecules or inhibition of negativeregulatory molecules of immune cell activation), inhibition of survivalsignals (e.g., growth factor or cytokine or hormone receptorantagonists), activation of death signals, and/or immune-mediatedcytotoxicity, such as through antibody dependent cellular cytotoxicity.Such IgSF conjugates can function through several mechanisms to prevent,reduce or eliminate tumor cells, such as to facilitate delivery ofconjugated effector moieties to the tumor target, such as throughreceptor-mediated endocytosis of the IgSF conjugate; or such conjugatescan recruit, bind, and/or activate immune cells (e.g. NK cells,monocytes/macrophages, dendritic cells, T cells, B cells).

In some embodiments, an IgSF conjugate, through its antibody targetingmoiety, will bind a cellular component of a tumor cell, tumorvasculature or tumor microenvironment, thereby modulating the immuneresponse (e.g., by activation of co-stimulatory molecules or inhibitionof negative regulatory molecules of immune cell activation). In someembodiments, such conjugates can recognize, bind, and/or modulate (e.g.inhibit or activate) immune cells (e.g. NK cells, monocytes/macrophages,dendritic cells, T cells, B cells).

Antibody targeting moieties of the invention include antibody fragmentsthat include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv) and fragments comprising either a VL or VH domain.Antigen-binding antibody fragments, including single-chain antibodies,may comprise the variable region(s) alone or in combination with theentirety or a portion of the following: hinge region, CH1, CH2, and CH3domains. Also included in the invention are antigen-binding fragmentsalso comprising any combination of variable region(s) with a hingeregion, CH1, CH2, and CH3 domains. Also included in the invention are Fcfragments, antigen-Fc fusion proteins, and Fc-targeting moietyconjugates or fusion products (Fc-peptide, Fc-aptamer). The antibodytargeting moieties of the invention may be from any animal originincluding birds and mammals. In one aspect, the antibody targetingmoieties are human, murine (e.g., mouse and rat), donkey, sheep, rabbit,goat, guinea pig, camel, horse, or chicken. Further, such antibodies maybe humanized versions of animal antibodies. The antibody targetingmoieties of the invention may be monospecific, bispecific, trispecific,or of greater multispecificity.

In various embodiments, an antibody/targeting moiety recruits, binds,and/or activates immune cells (e.g. NK cells, monocytes/macrophages,dendritic cells) via interactions between Fc (in antibodies) and Fcreceptors (on immune cells) and via the conjugated variant polypeptidesor immunomodulatory proteins provided herein. In some embodiments, anantibody/targeting moiety recognizes or binds a tumor agent via andlocalizes to the tumor cell the conjugated variant polypeptides orimmunomodulatory proteins provided herein to facilitate modulation ofimmune cells in the vicinity of the tumor.

Examples of antibodies which can be incorporated into IgSF conjugatesinclude but are not limited to antibodies such as Cetuximab (IMC-C225;Erbitux®), Trastuzumab (Herceptin®), Rituximab (Rituxan®; MabThera®),Bevacizumab (Avastin®), Alemtuzumab (Campath®; Campath-1H®;Mabcampath®), Panitumumab (ABX-EGF; Vectibix®), Ranibizumab (Lucentis®),Ibritumomab, Ibritumomab tiuxetan, (Zevalin®), Tositumomab, Iodine I 131Tositumomab (BEXXAR®), Catumaxomab (Removab®), Gemtuzumab, Gemtuzumabozogamicine (Mylotarg®), Abatacept (CTLA4-Ig; Orencia®), Belatacept(L104EA29YIg; LEA29Y; LEA), Ipilimumab (MDX-010; MDX-101), Tremelimumab(ticilimumab; CP-675,206), PRS-010, PRS-050, Aflibercept (VEGF Trap,AVE005), Volociximab (M200), F200, MORAb-009, SS1P (CAT-5001),Cixutumumab (IMC-A12), Matuzumab (EMD72000), Nimotuzumab (h-R3),Zalutumumab (HuMax-EGFR), Necitumumab IMC-11F8, mAb806/ch806, Sym004,mAb-425, Panorex@(17-1A) (murine monoclonal antibody); Panorex @(17-1A)(chimeric murine monoclonal antibody); IDEC- Y2B8 (murine, anti-CD2OMAb); BEC2 (anti-idiotypic MAb, mimics the GD epitope) (with BCG);Oncolym (Lym-1 monoclonal antibody); SMART MI95 Ab, humanized 13'I LYM-I(Oncolym), Ovarex (B43.13, anti-idiotypic mouse MAb); MDX-210 (humanizedanti-HER-2 bispecific antibody); 3622W94 MAb that binds to EGP40 (17-1A)pancarcinoma antigen on adenocarcinomas; Anti-VEGF, Zenapax (SMARTAnti-Tac (IL-2 receptor); SMART MI95 Ab, humanized Ab, humanized);MDX-210 (humanized anti- HER-2 bispecific antibody); MDX-447 (humanizedanti-EGF receptor bispecific antibody); NovoMAb-G2 (pancarcinomaspecific Ab); TNT (chimeric MAb to histone antigens); TNT (chimeric MAbto histone antigens); Gliomab-H (Monoclon s-Humanized Abs); GNI-250 Mab;EMD-72000 (chimeric-EGF antagonist); LymphoCide (humanized LL2antibody); and MDX-260 bispecific, targets GD-2, ANA Ab, SMART ID10 Ab,SMART ABL 364 Ab or ImmuRAIT-CEA. As illustrated by the forgoing list,it is conventional to make antibodies to a particular target epitope.

In some embodiments, the antibody targeting moiety is a full lengthantibody, or antigen-binding fragment thereof, containing an Fc domain.In some embodiments, the variant polypeptide or immunomodulatory proteinis conjugated to the Fc portion of the antibody targeting moiety, suchas by conjugation to the N-terminus of the Fc portion of the antibody.

In some embodiments, the vIgD is linked, directly or indirectly, to theN- or C-terminus of the light and/or heavy chain of the antibody. Insome embodiments, linkage can be via a peptide linker, such as anydescribed above. Various configurations can be constructed. FIG. 10A-10Cdepict exemplary configurations. In some embodiments, the antibodyconjugate can be produced by co-expression of the heavy and light chainof the antibody in a cell.

In one aspect of the invention, the targeting agent is an aptamermolecule. For example, in some embodiments, the aptamer is comprised ofnucleic acids that function as a targeting agent. In variousembodiments, an IgSF conjugate of the invention comprises an aptamerthat is specific for a molecule on a tumor cell, tumor vasculature,and/or a tumor microenvironment. In some embodiments, the aptamer itselfcan comprise a biologically active sequence, in addition to thetargeting module (sequence), wherein the biologically active sequencecan induce an immune response to the target cell. In other words, suchan aptamer molecule is a dual use agent. In some embodiments, an IgSFconjugate of the invention comprises conjugation of an aptamer to anantibody, wherein the aptamer and the antibody are specific for bindingto separate molecules on a tumor cell, tumor vasculature, tumormicroenvironment, and/or immune cells.

The term “aptamer” includes DNA, RNA or peptides that are selected basedon specific binding properties to a particular molecule. For example, anaptamer(s) can be selected for binding a particular gene or gene productin a tumor cell, tumor vasculature, tumor microenvironment, and/or animmune cell, as disclosed herein, where selection is made by methodsknown in the art and familiar to one of skill in the art.

In some aspects of the invention the targeting agent is a peptide. Forexample, the variant polypeptides or immunomodulatory proteins providedherein can be conjugated to a peptide which can bind with a component ofa cancer or tumor cells. Therefore, such IgSF conjugates of theinvention comprise peptide targeting agents which binds to a cellularcomponent of a tumor cell, tumor vasculature, and/or a component of atumor microenvironment. In some embodiments, targeting agent peptidescan be an antagonist or agonist of an integrin. Integrins, whichcomprise an alpha and a beta subunit, include numerous types well knownto a skilled artisan.

In one embodiment, the targeting agent is Vvβ3. Integrin Vvβ3 isexpressed on a variety of cells and has been shown to mediate severalbiologically relevant processes, including adhesion of osteoclasts tobone matrix, migration of vascular smooth muscle cells, andangiogenesis. Suitable targeting molecules for integrins include RGDpeptides or peptidomimetics as well as non-RGD peptides orpeptidomimetics (see, e.g., U.S. Pat. Nos. 5,767,071 and 5,780,426) forother integrins such as V4.βi (VLA-4), V4-P7 (see, e.g., U.S. Pat. No.6,365,619; Chang et al, Bioorganic & Medicinal Chem Lett, 12:159-163(2002); Lin et al., Bioorganic & Medicinal Chem Lett, 12:133-136(2002)), and the like.

In some embodiments, there is provided an IgSF conjugate comprising avariant polypeptide or immunomodulatory protein provided hereinconjugated with a therapeutic agent. In some embodiments, thetherapeutic agent includes, for example, daunomycin, doxorubicin,methotrexate, and vindesine (Rowland et al., Cancer Immunol. Immunother.21:183-187, 1986). In some embodiments, the therapeutic agent has anintracellular activity. In some embodiments, the IgSF conjugate isinternalized and the therapeutic agent is a cytotoxin that blocks theprotein synthesis of the cell, therein leading to cell death. In someembodiments, the therapeutic agent is a cytotoxin comprising apolypeptide having ribosome-inactivating activity including, forexample, gelonin, bouganin, saporin, ricin, ricin A chain, bryodin,diphtheria toxin, restrictocin, Pseudomonas exotoxin A and variantsthereof. In some embodiments, where the therapeutic agent is a cytotoxincomprising a polypeptide having a ribosome-inactivating activity, theIgSF conjugate must be internalized upon binding to the target cell inorder for the protein to be cytotoxic to the cells.

In some embodiments, there is provided an IgSF conjugate comprising avariant polypeptide or immunomodulatory protein provided hereinconjugated with a toxin. In some embodiments, the toxin includes, forexample, bacterial toxins such as diphtheria toxin, plant toxins such asricin, small molecule toxins such as geldanamycin (Mandler et al., J.Nat. Cancer Inst. 92(19):1573-1581 (2000); Mandler et al., Bioorganic &Med. Chem. Letters 10:1025- 1028 (2000); Mandler et al., BioconjugateChem. 13:786-791 (2002)), maytansinoids (EP 1391213; Liu et al., Proc.Natl. Acad. Sci. USA 93:8618-8623 (1996)), and calicheamicin (Lode etal., Cancer Res. 58:2928 (1998); Hinman et al., Cancer Res. 53:3336-3342(1993)). The toxins may exert their cytotoxic and cytostatic effects bymechanisms including tubulin binding, DNA binding, or topoisomeraseinhibition.

In some embodiments, there is provided an IgSF conjugate comprising avariant polypeptide or immunomodulatory protein provided hereinconjugated with a label, which can generate a detectable signal,indirectly or directly. These IgSF conjugates can be used for researchor diagnostic applications, such as for the in vivo detection of cancer.The label is preferably capable of producing, either directly orindirectly, a detectable signal. For example, the label may beradio-opaque or a radioisotope, such as 3H, 14C, 32P, 35S, 123I, 125I,131I; a fluorescent (fluorophore) or chemiluminescent (chromophore)compound, such as fluorescein isothiocyanate, rhodamine or luciferin; anenzyme, such as alkaline phosphatase, β-galactosidase or horseradishperoxidase; an imaging agent; or a metal ion. In some embodiments, thelabel is a radioactive atom for scintigraphic studies, for example 99Tcor 123I, or a spin label for nuclear magnetic resonance (NMR) imaging(also known as magnetic resonance imaging, MRI), such as zirconium-89,iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,oxygen-17, gadolinium, manganese or iron. Zirconium-89 may be complexedto various metal chelating agents and conjugated to antibodies, e.g.,for PET imaging (WO 2011/056983). In some embodiments, the IgSFconjugate is detectable indirectly. For example, a secondary antibodythat is specific for the IgSF conjugate and contains a detectable labelcan be used to detect the IgSF conjugate.

The IgSF conjugates may be prepared using any methods known in the art.See, e.g., WO 2009/067800, WO 2011/133886, and U.S. Patent ApplicationPublication No. 2014322129, incorporated by reference herein in theirentirety.

The variant polypeptides or immunomodulatory proteins of an IgSFconjugate may be “attached to” the effector moiety by any means by whichthe variant polypeptides or immunomodulatory proteins can be associatedwith, or linked to, the effector moiety. For example, the variantpolypeptides or immunomodulatory proteins of an IgSF conjugate may beattached to the effector moiety by chemical or recombinant means.Chemical means for preparing fusions or conjugates are known in the artand can be used to prepare the IgSF conjugate. The method used toconjugate the variant polypeptides or immunomodulatory proteins andeffector moiety must be capable of joining the variant polypeptides orimmunomodulatory proteins with the effector moiety without interferingwith the ability of the variant polypeptides or immunomodulatoryproteins to bind to their one or more counter structure ligands.

The variant polypeptides or immunomodulatory proteins of an IgSFconjugate may be linked indirectly to the effector moiety. For example,the variant polypeptides or immunomodulatory proteins of an IgSFconjugate may be directly linked to a liposome containing the effectormoiety of one of several types. The effector moiety(s) and/or thevariant polypeptides or immunomodulatory proteins may also be bound to asolid surface.

In some embodiments, the variant polypeptides or immunomodulatoryproteins of an IgSF conjugate and the effector moiety are both proteinsand can be conjugated using techniques well known in the art. There areseveral hundred crosslinkers available that can conjugate two proteins.(See for example “Chemistry of Protein Conjugation and Crosslinking,”1991, Shans Wong, CRC Press, Ann Arbor). The crosslinker is generallychosen based on the reactive functional groups available or inserted onthe variant polypeptides or immunomodulatory proteins and/or effectormoiety. In addition, if there are no reactive groups, a photoactivatiblecrosslinker can be used. In certain instances, it may be desirable toinclude a spacer between the variant polypeptides or immunomodulatoryproteins and the effector moiety. Crosslinking agents known to the artinclude the homobifunctional agents: glutaraldehyde, dimethyladipimidateand Bis(diazobenzidine) and the heterobifunctional agents: mMaleimidobenzoyl-N-Hydroxysuccinimide and Sulfo-mMaleimidobenzoyl-N-Hydroxysuccinimide.

In some embodiments, the variant polypeptides or immunomodulatoryproteins of an IgSF conjugate may be engineered with specific residuesfor chemical attachment of the effector moiety. Specific residues usedfor chemical attachment of molecule known to the art include lysine andcysteine. The crosslinker is chosen based on the reactive functionalgroups inserted on the variant polypeptides or immunomodulatoryproteins, and available on the effector moiety.

An IgSF conjugate may also be prepared using recombinant DNA techniques.In such a case a DNA sequence encoding the variant polypeptides orimmunomodulatory proteins is fused to a DNA sequence encoding theeffector moiety, resulting in a chimeric DNA molecule. The chimeric DNAsequence is transfected into a host cell that expresses the fusionprotein. The fusion protein can be recovered from the cell culture andpurified using techniques known in the art.

Examples of attaching an effector moiety, which is a label, to thevariant polypeptides or immunomodulatory proteins include the methodsdescribed in Hunter, et al., Nature 144:945 (1962); David, et al.,Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219(1981); Nygren, J. Histochem. and Cytochem. 30:407 (1982); Wensel andMeares, Radioimmunoimaging And Radioimmunotherapy, Elsevier, N.Y.(1983); and Colcher et al., “Use Of Monoclonal Antibodies AsRadiopharmaceuticals For The Localization Of Human Carcinoma XenograftsIn Athymic Mice”, Meth. Enzymol., 121:802-16 (1986).

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as 99Tc or 123I, 186Re, 188Re and 111In can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al., Biochem. Biophys.Res. Commun. 80:49-57 (1978)) can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the variant polypeptides or immunomodulatory proteins anda cytotoxic agent may be made using a variety of bifunctional proteincoupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate(SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate(SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters(such as dimethyl adipimidate HCI), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azidocompounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine),diisocyanates (such as toluene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Forexample, a ricin immunotoxin can be prepared as described in Vitetta etal., Science 238:1098 (1987). Carbon-14-labeled1-isothiocyanatobenzyl-3-methyldiethylene tnaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the antibody. See, e.g., WO94/11026. The linker maybe a “cleavable linker” facilitating release of the cytotoxic drug inthe cell. For example, an acid-labile linker, peptidase-sensitivelinker, photolabile linker, dimethyl linker or disulfide-containinglinker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No.5,208,020) may be used.

The IgSF conjugates of the invention expressly contemplate, but are notlimited to, drug conjugates prepared with cross-linker reagents: BMPS,EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH,sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC,and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) whichare commercially available (e.g., from Pierce Biotechnology, Inc.,Rockford, Ill., U.S.A). See pages 467-498, 2003-2004 ApplicationsHandbook and Catalog.

D. Transmembrane and Secretable Immunomodulatory Proteins and EngineeredCells

Provided herein are engineered cells which express the immunomodulatoryvariant ICOSL polypeptides (alternatively, “engineered cells”). In someembodiments, the expressed immunomodulatory variant ICOSL polypeptide isa transmembrane proteins and is surface expressed. In some embodiments,the expressed immunomodulatory variant ICOSL polypeptide is expressedand secreted from the cell.

1. Transmembrane Immunomodulatory Proteins

In some embodiments, an immunomodulatory polypeptide comprising avariant ICOSL can be a membrane bound protein. As described in moredetail below, the immunomodulatory polypeptide can be a transmembraneimmunomodulatory polypeptide comprising a variant ICOSL in which iscontained: an ectodomain containing at least one affinity modified IgSFdomain (IgV or IgC), a transmembrane domain and, optionally, acytoplasmic domain. In some embodiments, the transmembraneimmunomodulatory protein can be expressed on the surface of an immunecell, such as a mammalian cell, including on the surface of a lymphocyte(e.g. T cell or NK cell) or antigen presenting cell. In someembodiments, the transmembrane immunomodulatory protein is expressed onthe surface of a mammalian T-cell, including such T-cells as: a T helpercell, a cytotoxic T-cell (alternatively, cytotoxic T lymphocyte or CTL),a natural killer T-cell, a regulatory T-cell, a memory T-cell, or agamma delta T-cell. In some embodiments, the mammalian cell is anantigen presenting cell (APC). Typically, but not exclusively, theectodomain (alternatively, “extracellular domain”) of comprises the oneor more amino acid variations (e.g. amino acid substitutions) of thevariant ICOSL of the invention. Thus, for example, in some embodiments atransmembrane protein will comprise an ectodomain that comprises one ormore amino acid substitutions of a variant ICOSL of the invention.

In some embodiments, the engineered cells express variant ICOSLpolypeptides that are transmembrane immunomodulatory polypeptides (TIN)that can be a membrane protein such as a transmembrane protein. Intypical embodiments, the ectodomain of a membrane protein comprises anextracellular domain or IgSF domain thereof of a variant ICOSL providedherein in which is contained one or more amino acid substitutions in atleast one IgSF domain as described. The transmembrane immunomodulatoryproteins provided herein further contain a transmembrane domain linkedto the ectodomain. In some embodiments, the transmembrane domain resultsin an encoded protein for cell surface expression on a cell. In someembodiments, the transmembrane domain is linked directly to theectodomain. In some embodiments, the transmembrane domain is linkedindirectly to the ectodomain via one or more linkers or spacers. In someembodiments, the transmembrane domain contains predominantly hydrophobicamino acid residues, such as leucine and valine.

In some embodiments, a full length transmembrane anchor domain can beused to ensure that the TIPs will be expressed on the surface of theengineered cell, such as engineered T cell. Conveniently, this could befrom a particular native protein that is being affinity modified (e.g.ICOSL or other native IgSF protein), and simply fused to the sequence ofthe first membrane proximal domain in a similar fashion as the nativeIgSF protein (e.g. ICOSL). In some embodiments, the transmembraneimmunomodulatory protein comprises a transmembrane domain of thecorresponding wild-type or unmodified IgSF member, such as atransmembrane domain contained in the sequence of amino acids set forthin SEQ ID NO:5 (Table 2). In some embodiments, the membrane bound formcomprises a transmembrane domain of the corresponding wild-type orunmodified polypeptide, such as corresponding to residues 257-277 of SEQID NO:5.

In some embodiments, the transmembrane domain is a non-nativetransmembrane domain that is not the transmembrane domain of nativeICOSL. In some embodiments, the transmembrane domain is derived from atransmembrane domain from another non-ICOSL family member polypeptidethat is a membrane-bound or is a transmembrane protein. In someembodiments, a transmembrane anchor domain from another protein on Tcells can be used. In some embodiments, the transmembrane domain isderived from CD8. In some embodiments, the transmembrane domain canfurther contain an extracellular portion of CD8 that serves as a spacerdomain. An exemplary CD8 derived transmembrane domain is set forth inSEQ ID NO: 246 or 483 or a portion thereof containing the CD8transmembrane domain. In some embodiments, the transmembrane domain is asynthetic transmembrane domain.

In some embodiments, the transmembrane immunomodulatory protein furthercontains an endodomain, such as a cytoplasmic signaling domain, linkedto the transmembrane domain. In some embodiments, the cytoplasmicsignaling domain induces cell signaling. In some embodiments, theendodomain of the transmembrane immunomodulatory protein comprises thecytoplasmic domain of the corresponding wild-type or unmodifiedpolypeptide, such as a cytoplasmic domain contained in the sequence ofamino acids set forth in SEQ ID NO:5 (see Table 2).

In some embodiments, a provided transmembrane immunomodulatory proteinthat is or comprises a variant ICOSL comprises a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 257 andcontains an ectodomain comprising at least one affinity-modified ICOSLIgSF domain as described and a transmembrane domain. In someembodiments, the transmembrane immunomodulatory protein contains any oneor more amino acid substitutions in an IgSF domain (e.g. IgV domain) asdescribed, including any set forth in Table 1. In some embodiments, thetransmembrane immunomodulatory protein can further comprise acytoplasmic domain as described. In some embodiments, the transmembraneimmunomodulatory protein can further contain a signal peptide. In someembodiments, the signal peptide is the native signal peptide ofwild-type IgSF member, such as contained in the sequence of amino acidsset forth in SEQ ID NO:5 (see e.g. Table 2).

In some embodiments, provided are transmembrane immunomodulatoryproteins comprising the amino acid substitutionsE16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R, N52H/N57Y/Q100R, orN52H/N57Y/Q100P. In some embodiments, the provided transmembraneimmunomodulatory protein is or comprises a variant ICOSL comprising thesequence of amino acids set forth in SEQ ID NO:257, but in which iscontained amino substitutionsE16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R, N52H/N57Y/Q100R, orN52H/N57Y/Q100P at corresponding positions in SEQ ID NO:257, or asequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity toSEQ ID NO: 257 and contains the amino acid substitutionsE16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R, N52H/N57Y/Q100R, orN52H/N57Y/Q100P.

In some embodiments, provided are transmembrane immunomodulatoryproteins comprising the sequence of amino acids set forth in SEQ ID NOS:496 or 497 (each containing the amino acid substitution N52D), SEQ IDNOS: 498 or 499 (each containing the amino acid substitutionsN52H/N57Y/Q100P), SEQ ID NOS: 500 or 501 (each containing the amino acidsubstitutions E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R) or SEQID NOS: 502 or 503 (each containing the amino acid substitutionsN52H/N57Y/Q100R), or a sequence of amino acids that comprises at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to any of SEQ ID NOS: 495-503 and that containsthe indicated amino acid substitutions. In some embodiments, whenexpressed in an engineered cell, such transmembrane immunomodulatoryproteins are expressed on the surface of the cell.

Also provided is a nucleic acid molecule encoding such transmembraneimmunomodulatory proteins. In some embodiments, a nucleic acid moleculeencoding a transmembrane immunomodulatory protein comprises a nucleotidesequence that encodes a sequence of amino acids that exhibits at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to SEQ ID NOS: 257 and contains an ectodomaincomprising at least one affinity-modified IgSF domain as described, atransmembrane domain and, optionally, a cytoplasmic domain. In someembodiments, the nucleic acid molecule can further comprise a sequenceof nucleotides encoding a signal peptide. In some embodiments, thesignal peptide is the native signal peptide of the correspondingwild-type IgSF member (see e.g. Table 2).

Exemplary of a transmembrane immunomodulatory protein is a ICOSL TIPcomprising i) the sequence of amino acids set forth in SEQ ID NO:383 orii) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to SEQ ID NO:243 and that comprises the affinity-modifieddomain contained in SEQ ID NO:243 or the amino acid substitutionstherein. Also provided is i) a sequence of nucleotides set forth in SEQID NO:244, ii) a sequence that exhibits at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to SEQ ID NO: 244 and that encodes a TIP that comprises theaffinity-modified domain of SEQ ID NO:243, or iii) a sequence of i) orii) having degenerate codons.

In some embodiments, provided are CAR-related transmembraneimmunomodulatory proteins in which the endodomain of a transmembraneimmunomodulatory protein comprises a cytoplasmic signaling domain thatcomprises at least one ITAM (immunoreceptor tyrosine-based activationmotif)-containing signaling domain. ITAM is a conserved motif found in anumber of protein signaling domains involved in signal transduction ofimmune cells, including in the CD3-zeta chain (“CD3-z”) involved inT-cell receptor signal transduction. In some embodiments, the endodomaincomprises at CD3-zeta signaling domain. In some embodiments, theCD3-zeta signaling domain comprises the sequence of amino acids setforth in SEQ ID NO: 243 or a sequence of amino acids that exhibits atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% to SEQ ID NO:247 and retains the activity of T cellsignaling. In some embodiments, the endodomain of a CAR-relatedtransmembrane immunomodulatory protein can further comprise acostimulatory signaling domain to further modulate immunomodulatoryresponses of the T-cell. In some embodiments, the costimulatorysignaling domain is CD28, ICOS, 41BB or OX40. In some embodiments, thecostimulatory signaling domain is a derived from CD28 or 4-1BB andcomprises the sequence of amino acids set forth in any of SEQ ID NOS:484-487 or a sequence of amino acids that exhibits at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to SEQID NO:484-487 and retains the activity of T cell costimulatorysignaling. In some embodiments, the provided CAR-related transmembraneimmunomodulatory proteins have features of CARs to stimulate T cellsignaling upon binding of an affinity modified IgSF domain to a cognatebinding partner or counter structure. In some embodiments, upon specificbinding by the affinity-modified IgSF domain to its counter structurecan lead to changes in the immunological activity of the T-cell activityas reflected by changes in cytotoxicity, proliferation or cytokineproduction.

In some embodiments, the transmembrane immunomodulatory protein does notcontain an endodomain capable of mediating cytoplasmic signaling. Insome embodiments, the transmembrane immunomodulatory protein lacks thesignal transduction mechanism of the wild-type or unmodified polypeptideand therefore does not itself induce cell signaling. In someembodiments, the transmembrane immunomodulatory protein lacks anintracellular (cytoplasmic) domain or a portion of the intracellulardomain of the corresponding wild-type or unmodified polypeptide, such asa cytoplasmic signaling domain contained in the sequence of amino acidsset forth in SEQ ID NO:5 (see Table 2). In some embodiments, thetransmembrane immunomodulatory protein does not contain an ITIM(immunoreceptor tyrosine-based inhibition motif), such as contained incertain inhibitory receptors, including inhibitory receptors of the IgSFfamily (e.g. PD-1 or TIGIT). Thus, in some embodiments, thetransmembrane immunomodulatory protein only contains the ectodomain andthe transmembrane domain, such as any as described.

2. Secreted Immunomodulatory Proteins and Engineered Cells

In some embodiments, the ICOSL variant immunomodulatory polypeptidecontaining any one or more of the amino acid mutations as describedherein, is secretable, such as when expressed from a cell. Such avariant ICOSL immunomodulatory protein does not comprise a transmembranedomain. In some embodiments, the variant ICOSL immunomodulatory proteinis not conjugated to a half-life extending moiety (such as an Fc domainor a multermization domain). In some embodiments, the variant ICOSLimmunomodulatory protein comprises a signal peptide, e.g. an antibodysignal peptide or other efficient signal sequence to get domains outsideof cell. When the immunomodulatory protein comprises a signal peptideand is expressed by an engineered cell, the signal peptide causes theimmunomodulatory protein to be secreted by the engineered cell.Generally, the signal peptide, or a portion of the signal peptide, iscleaved from the immunomodulatory protein with secretion. Theimmunomodulatory protein can be encoded by a nucleic acid (which can bepart of an expression vector). In some embodiments, the immunomodulatoryprotein is expressed and secreted by a cell (such as an immune cell, forexample a primary immune cell).

Thus, in some embodiments, there are provided variant ICOSLimmunomodulatory proteins that further comprise a signal peptide. Insome embodiments, provided herein is a nucleic acid molecule encodingthe variant ICOSL immunomodulatory protein operably connected to asecretion sequence encoding the signal peptide.

A signal peptide is a sequence on the N-terminus of an immunomodulatoryprotein that signals secretion of the immunomodulatory protein from acell. In some embodiments, the signal peptide is about 5 to about 40amino acids in length (such as about 5 to about 7, about 7 to about 10,about 10 to about 15, about 15 to about 20, about 20 to about 25, orabout 25 to about 30, about 30 to about 35, or about 35 to about 40amino acids in length).

In some embodiments, the signal peptide is a native signal peptide fromthe corresponding wild-type ICOSL (see Table 6). In some embodiments,the signal peptide is a non-native signal peptide. For example, in someembodiments, the non-native signal peptide is a mutant native signalpeptide from the corresponding wild-type ICOSL, and can include one ormore (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) substitutionsinsertions or deletions. In some embodiments, the non-native signalpeptide is a signal peptide or mutant thereof of a family member fromthe same IgSF family as the wild-type IgSF family member. In someembodiments, the non-native signal peptide is a signal peptide or mutantthereof from an IgSF family member from a different IgSF family than thewild-type IgSF family member. In some embodiments, the signal peptide isa signal peptide or mutant thereof from a non-IgSF protein family, suchas a signal peptide from an immunoglobulin (such as IgG heavy chain orIgG-kappa light chain), a cytokine (such as interleukin-2 (IL-2), orCD33), a serum albumin protein (e.g. HSA or albumin), a human azurocidinpreprotein signal sequence, a luciferase, a trypsinogen (e.g.chumotrypsinogen or trypsinogen) or other signal peptide able toefficiently secrete a protein from a cell. Exemplary signal peptidesinclude any described in the Table 6.

TABLE 6 Exemplary Signal Peptides SEQ ID NO Signal PeptidePeptide Sequence SEQ ID NO: 346 HSA signal peptide MKWVTFISLLFLFSSAYSSEQ ID NO: 347 Ig kappa light chain MDMRAPAGIFGFLLVLFPGYRSSEQ ID NO: 348 human azurocidin preprotein MTRLTVLALLAGLLASSRAsignal sequence SEQ ID NO: 349 IgG heavy chain signal peptideMELGLSWIFLLAILKGVQC SEQ ID NO: 350 IgG heavy chain signal peptideMELGLRWVFLVAILEGVQC SEQ ID NO: 351 IgG heavy chain signal peptideMKHLWFFLLLVAAPRWVLS SEQ ID NO: 352 IgG heavy chain signal peptideMDWTWRILFLVAAATGAHS SEQ ID NO: 353 IgG heavy chain signal peptideMDWTWRFLFVVAAATGVQS SEQ ID NO: 354 IgG heavy chain signal peptideMEFGLSWLFLVAILKGVQC SEQ ID NO: 355 IgG heavy chain signal peptideMEFGLSWVFLVALFRGVQC SEQ ID NO: 356 IgG heavy chain signal peptideMDLLHKNMKHLWFFLLLVAAPRWVLS SEQ ID NO: 357 IgG Kappa light chain signalMDMRVPAQLLGLLLLWLSGARC sequence SEQ ID NO: 358IgG Kappa light chain signal MKYLLPTAAAGLLLLAAQPAMA sequenceSEQ ID NO: 359 Gaussia luciferase MGVKVLFALICIAVAEA SEQ ID NO: 360Human albumin MKWVTFISLLFLFSSAYS SEQ ID NO: 361 Human chymotrypsinogenMAFLWLLSCWALLGTTFG SEQ ID NO: 362 Human interleukin-2 MQLLSCIALILALVSEQ ID NO: 363 Human trypsinogen-2 MNLLLILTFVAAAVA

In some embodiments of a secretable variant ICOSL immunomodulatoryprotein, the immunomodulatory protein comprises a signal peptide whenexpressed, and the signal peptide (or a portion thereof) is cleaved fromthe immunomodulatory protein upon secretion.

In some embodiments, the engineered cells expresses a variant ICOSLpolypeptides that are secreted from the cell. In some embodiments, sucha variant ICOSL polypeptide is encoded by a nucleic acid moleculeencoding an immunomodulatory protein under the operable control of asignal sequence for secretion. In some embodiments, the encodedimmunomodulatory protein is secreted when expressed from a cell. In someembodiments, the immunomodulatory protein encoded by the nucleic acidmolecule does not comprise a transmembrane domain. In some embodiments,the immunomodulatory protein encoded by the nucleic acid molecule doesnot comprise a half-life extending moiety (such as an Fc domain or amultimerization domain). In some embodiments, the immunomodulatoryprotein encoded by the nucleic acid molecule comprises a signal peptide.In some embodiments, a nucleic acid of the invention further comprisesnucleotide sequence that encodes a secretory or signal peptide operablylinked to the nucleic acid encoding the immunomodulatory protein,thereby allowing for secretion of the immunomodulatory protein 3. Cellsand Engineering Cells

Provided herein are engineered cells expressing any of the providedimmunomodulatory polypeptide. In some embodiments, the engineered cellsexpress on their surface any of the provided transmembraneimmunomodulatory polypeptides. In some embodiments, the engineered cellsexpress and are capable of or are able to secrete the immunomodulatoryprotein from the cells under conditions suitable for secretion of theprotein. In some embodiments, the immunomodulatory protein is expressedon a lymphocyte such as a tumor infiltrating lymphocyte (TIL), T-cell orNK cell, or on a myeloid cell. In some embodiments, the engineered cellsare antigen presenting cells (APCs). In some embodiments, the engineeredcells are engineered mammalian T-cells or engineered mammalian antigenpresenting cells (APCs). In some embodiments, the engineered T-cells orAPCs are human or murine cells.

In some embodiments, engineered T-cells include, but are not limited to,T helper cell, cytotoxic T-cell (alternatively, cytotoxic T lymphocyteor CTL), natural killer T-cell, regulatory T-cell, memory T-cell, orgamma delta T-cell. In some embodiments, the engineered T cells are CD4+or CD8+. In addition to the signal of the MHC, engineered T-cells alsorequire a co-stimulatory signal which in some embodiments is provided bya variant ICOSL transmembrane immunomodulatory polypeptide expressed inmembrane bound form as discussed previously.

In some embodiments, the engineered APCs include, for example, MHC IIexpressing APCs such as macrophages, B cells, and dendritic cells, aswell as artificial APCs (aAPCs) including both cellular and acellular(e.g., biodegradable polymeric microparticles) aAPCs. Artificial APCs(aAPCs) are synthetic versions of APCs that can act in a similar mannerto APCs in that they present antigens to T-cells as well as activatethem. Antigen presentation is performed by the MHC (Class I or ClassII). In some embodiments, in engineered APCs such as aAPCs, the antigenthat is loaded onto the MHC is, in some embodiments, a tumor specificantigen or a tumor associated antigen. The antigen loaded onto the MHCis recognized by a T-cell receptor (TCR) of a T cell, which, in somecases, can express ICOS, CD28, or other molecule recognized by thevariant ICOSL polypeptides provided herein. Materials which can be usedto engineer an aAPC include: poly (glycolic acid),poly(lactic-co-glycolic acid), iron-oxide, liposomes, lipid bilayers,sepharose, and polystyrene.

In some embodiments, an immunomodulatory protein, such as atransmembrane immunomodulatory protein or a secretable immunomodulatoryprotein, provided herein is co-expressed or engineered into a cell thatexpresses an antigen-binding receptor, such as a recombinant receptor,such as a chimeric antigen receptor (CAR) or T cell receptor (TCR). Insome embodiments, the engineered cell, such as an engineered T cell,recognizes a desired antigen associated with cancer, inflammatory andautoimmune disorders, or a viral infection. In specific embodiments, theantigen-binding receptor contains an antigen-binding moiety thatspecifically binds a tumor specific antigen or a tumor associatedantigen. In some embodiments, the engineered T-cell is a CAR (chimericantigen receptor) T-cell that contains an antigen-binding domain (e.g.scFv) that specifically binds to an antigen, such as a tumor specificantigen or tumor associated antigen. In some embodiments, theantigen-binding domain (e.g. scFv) is specific for CD19. Exemplary of aCAR is an anti-CD19 CAR, such as a CAR containing an anti-CD19 scFv setforth in SEQ ID NO:482 or SEQ ID NO:245. In some embodiments, the TIPprotein is expressed in an engineered T-cell receptor cell or andengineered chimeric antigen receptor cell. In such embodiments, theengineered cell co-expresses the TIP and the CAR or TCR.

In some embodiments, the CAR further contains a spacer or hinge, atransmembrane domain, and an intracellular signaling domain (endodomain)comprising an ITAM signaling domain, such as a CD3zeta signaling domain.In some embodiments, the CAR futher includes a costimulatory signalingdomain. In some embodiments, the spacer or hinge is present between theantigen-binding domain and the transmembrane domain, such as is betweenthe antigen-binding domain and plasma membrane when expressed on a cell.In some embodiments, the spacer or hinge is derived from IgG subclass(such as IgG1 and IgG4, IgD or CD8 (see e.g., Qin et al. (2017) J.Hematol. Oncol., 10:68). In some embodiments, the spacer or hinge isderived from IgG1.

In some embodiments, the spacer and transmembrane domain are the hingeand transmembrane domain derived from CD8, such as set forth in SEQ IDNO: 246 or 483 or a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to SEQ ID NO:246 or 483. In some embodiments, theendodomain comprises at CD3-zeta signaling domain. In some embodiments,the CD3-zeta signaling domain comprises the sequence of amino acids setforth in SEQ ID NO: 243 or a sequence of amino acids that exhibits atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% to SEQ ID NO:247 and retains the activity of T cellsignaling. In some embodiments, the endodomain of a CAR-relatedtransmembrane immunomodulatory protein can further comprise acostimulatory signaling domain to further modulate immunomodulatoryresponses of the T-cell. In some embodiments, the costimulatorysignaling domain is CD28, ICOS, 41BB or OX40. In some embodiments, thecostimulatory signaling domain is a derived from CD28 or 4-1BB andcomprises the sequence of amino acids set forth in any of SEQ ID NOS:484-487 or a sequence of amino acids that exhibits at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to SEQID NO:484-487 and retains the activity of T cell costimulatorysignaling.

In another embodiment, the engineered T-cell possesses a TCR, includinga recombinant or engineered TCR. In some embodiments, the TCR can be anative TCR. Those of skill in the art will recognize that generallynative mammalian T-cell receptors comprise an alpha and a beta chain (ora gamma and a delta chain) involved in antigen specific recognition andbinding. In some embodiments, the TCR is an engineered TCR that ismodified. In some embodiments, the TCR of an engineered T-cellspecifically binds to a tumor associated or tumor specific antigenpresented by an APC.

In some embodiments, the immunomodulatory polypeptides, such astransmembrane immunomodulatory polypeptides or secretableimmunomodulatory polypeptides, can be incorporated into engineeredcells, such as engineered T cells or engineered APCs, by a variety ofstrategies such as those employed for recombinant host cells. A varietyof methods to introduce a DNA construct into primary T cells are knownin the art. In some embodiments, viral transduction or plasmidelectroporation are employed. In typical embodiments, the nucleic acidmolecule encoding the immunomodulatory protein, or the expressionvector, comprises a signal peptide that localizes the expressedtransmembrane immunomodulatory proteins to the cellular membrane or forsecretion. In some embodiments, a nucleic acid encoding a transmembraneimmunomodulatory proteins of the invention is sub-cloned into a viralvector, such as a retroviral vector, which allows expression in the hostmammalian cell. The expression vector can be introduced into a mammalianhost cell and, under host cell culture conditions, the immunomodulatoryprotein is expressed on the surface or is secreted.

In an exemplary example, primary T-cells can be purified ex vivo (CD4cells or CD8 cells or both) and stimulated with an activation protocolconsisting of various TCR/CD28 agonists, such as anti-CD3/anti-CD28coated beads. After a 2 or 3 day activation process, a recombinantexpression vector containing an immunomodulatory polypeptide can bestably introduced into the primary T cells through art standardlentiviral or retroviral transduction protocols or plasmidelectroporation strategies. Cells can be monitored for immunomodulatorypolypeptide expression by, for example, flow cytometry usinganti-epitope tag or antibodies that cross-react with native parentalmolecule and polypeptides comprising variant ICOSL. T-cells that expressthe immunomodulatory polypeptide can be enriched through sorting withanti-epitope tag antibodies or enriched for high or low expressiondepending on the application.

Upon immunomodulatory polypeptide expression the engineered T-cell canbe assayed for appropriate function by a variety of means. Theengineered CAR or TCR co-expression can be validated to show that thispart of the engineered T cell was not significantly impacted by theexpression of the immunomodulatory protein. Once validated, standard invitro cytotoxicity, proliferation, or cytokine assays (e.g., IFN-gammaexpression) can be used to assess the function of engineered T-cells.Exemplary standard endpoints are percent lysis of the tumor line,proliferation of the engineered T-cell, or IFN-gamma protein expressionin culture supernatants. An engineered construct which results instatistically significant increased lysis of tumor line, increasedproliferation of the engineered T-cell, or increased IFN-gammaexpression over the control construct can be selected for. Additionally,non-engineered, such as native primary or endogenous T-cells could alsobe incorporated into the same in vitro assay to measure the ability ofthe immunomodulatory polypeptide construct expressed on the engineeredcells, such as engineered T-cells, to modulate activity, including, insome cases, to activate and generate effector function in bystander,native T-cells. Increased expression of activation markers such as CD69,CD44, or CD62L could be monitored on endogenous T cells, and increasedproliferation and/or cytokine production could indicate desired activityof the immunomodulatory protein expressed on the engineered T cells.

In some embodiments, the similar assays can be used to compare thefunction of engineered T cells containing the CAR or TCR alone to thosecontaining the CAR or TCR and a TIP construct. Typically, these in vitroassays are performed by plating various ratios of the engineered T celland a “tumor” cell line containing the cognate CAR or TCR antigentogether in culture. Standard endpoints are percent lysis of the tumorline, proliferation of the engineered T cell, or IFN-gamma production inculture supernatants. An engineered immunomodulatory protein whichresulted in statistically significant increased lysis of tumor line,increased proliferation of the engineered T cell, or increased IFN-gammaproduction over the same TCR or CAR construct alone can be selected for.Engineered human T cells can be analyzed in immunocompromised mice, likethe NSG strain, which lacks mouse T, NK and B cells. Engineered human Tcells in which the CAR or TCR binds a target counter-structure on thexenograft and is co-expressed with the TIP affinity modified IgSF domaincan be adoptively transferred in vivo at different cell numbers andratios compared to the xenograft. For example, engraftment of CD19+leukemia tumor lines containing a luciferase/GFP vector can be monitoredthrough bioluminescence or ex vivo by flow cytometry. In a commonembodiment, the xenograft is introduced into the murine model, followedby the engineered T cells several days later. Engineered T cellscontaining the immunomodulatory protein can be assayed for increasedsurvival, tumor clearance, or expanded engineered T cells numbersrelative to engineered T cells containing the CAR or TCR alone. As inthe in vitro assay, endogenous, native (i.e., non-engineered) human Tcells could be co-adoptively transferred to look for successful epitopespreading in that population, resulting in better survival or tumorclearance.

E. Infectious Agents Expressing Variant Polypeptides andImmunomodulatory Proteins

Also provided are infectious agents that contain nucleic acids encodingany of the variant polypeptides, such as ICOSL vIgD polypeptides,including secretable or transmembrane immunomodulatory proteinsdescribed herein. In some embodiments, such infectious agents candeliver the nucleic acids encoding the variant immunomodulatorypolypeptides described herein, such as ICOSL vIgD polypeptides, to atarget cell in a subject, e.g., immune cell and/or antigen-presentingcell (APC) or tumor cell in a subject. Also provided are nucleic acidscontained in such infectious agents, and/or nucleic acids for generationor modification of such infectious agents, such as vectors and/orplasmids, and compositions containing such infectious agents.

In some embodiments, the infectious agent is a microorganism or amicrobe. In some embodiments, the infectious agent is a virus or abacterium. In some embodiments, the infectious agent is a virus. In someembodiments, the infectious agent is a bacterium. In some embodiments,such infectious agents can deliver nucleic acid sequences encoding anyof the variant polypeptides, such as ICOSL vIgD polypeptides, includingsecretable or transmembrane immunomodulatory proteins, described herein.Thus, in some embodiments, the cell in a subject that is infected orcontacted by the infectious agents can be rendered to express on thecell surface or secrete, the variant immunomodulatory polypeptides. Insome embodiments, the infectious agent can also deliver one or moreother therapeutics or nucleic acids encoding other therapeutics to thecell and/or to an environment within the subject. In some embodiments,other therapeutics that can be delivered by the infectious agentsinclude cytokines or other immunomodulatory molecules.

In some embodiments, the infectious agent, e.g., virus or bacteria,contains nucleic acid sequences that encode any of the variantpolypeptides, such as ICOSL vIgD polypeptides, including secretable ortransmembrane immunomodulatory proteins, described herein, and by virtueof contact and/or infection of a cell in the subject, the cell expressesthe variant polypeptides, such as ICOSL vIgD polypeptides, includingsecretable or transmembrane immunomodulatory proteins, encoded by thenucleic acid sequences contained in the infectious agent. In someembodiments, the infectious agent can be administered to the subject. Insome embodiments, the infectious agent can be introduced to cells fromthe subject ex vivo.

In some embodiments, the variant polypeptides, such as ICOSL vIgDpolypeptides, including transmembrane immunomodulatory proteins,expressed by the cell infected by the infectious agent is atransmembrane protein and is surface expressed. In some embodiments, thevariant polypeptides, such as ICOSL vIgD polypeptides, includingsecretable immunomodulatory proteins, expressed by the cell infected bythe infectious agent is expressed and secreted from the cell. Thetransmembrane immunomodulatory protein or secreted immunomodulatoryprotein can be any described herein.

In some embodiments, the cells in the subject that are targeted by theinfectious agent include a tumor cell, an immune cell, and/or anantigen-presenting cell (APC). In some embodiments, the infectious agenttargets a cell in the tumor microenvironment (TME). In some embodiments,the infectious agent delivers the nucleic acids encoding the variantpolypeptides, such as ICOSL vIgD polypeptides, including secretable ortransmembrane immunomodulatory proteins, to an appropriate cell (forexample, an APC, such as a cell that displays a peptide/MHC complex onits cell surface, such as a dendritic cell) or tissue (e.g., lymphoidtissue) that will induce and/or augment the desired effect, e.g.,immunomodulation and/or a specific cell-medicated immune response, e.g.,CD4 and/or CD8 T cell response, which CD8 T cell response may include acytotoxic T cell (CTL) response. In some embodiments, the infectiousagent targets an APC, such as a dendritic cell (DC). In someembodiments, the nucleic acid molecule delivered by the infectiousagents described herein include appropriate nucleic acid sequencesnecessary for the expression of the operably linked coding sequencesencoding the variant immunomodulatory polypeptides, in a particulartarget cell, e.g., regulatory elements such as promoters.

In some embodiments, the infectious agent that contains nucleic acidsequences encoding the immunomodulatory polypeptides can also containnucleic acid sequences that encode one or more additional gene products,e.g., cytokines, prodrug converting enzymes, cytotoxins and/ordetectable gene products. For example, in some embodiments, theinfectious agent is an oncolytic virus and the virus can include nucleicacid sequences encoding additional therapeutic gene products (see, e.g.,Kim et al., (2009) Nat Rev Cancer 9:64-71; Garcia-Aragoncillo et al.,(2010) Curr Opin Mol Ther 12:403-411; see U.S. Pat. Nos. 7,588,767,7,588,771, 7,662,398 and 7,754,221 and U.S. Pat. Publ. Nos.2007/0202572, 2007/0212727, 2010/0062016, 2009/0098529, 2009/0053244,2009/0155287, 2009/0117034, 2010/0233078, 2009/0162288, 2010/0196325,2009/0136917 and 2011/0064650. In some embodiments, the additional geneproduct can be a therapeutic gene product that can result in death ofthe target cell (e.g., tumor cell) or gene products that can augment orboost or regulate an immune response (e.g., cytokine). Exemplary geneproducts also include among an anticancer agent, an anti-metastaticagent, an antiangiogenic agent, an immunomodulatory molecule, an immunecheckpoint inhibitor, an antibody, a cytokine, a growth factor, anantigen, a cytotoxic gene product, a pro-apoptotic gene product, ananti-apoptotic gene product, a cell matrix degradative gene, genes fortissue regeneration and reprogramming human somatic cells topluripotency, and other genes described herein or known to one of skillin the art. In some embodiments, the additional gene product isGranulocyte-macrophage colony-stimulating factor (GM-CSF).

1. Viruses

In some embodiments, the infectious agent is a virus. In someembodiments, the infectious agent is an oncolytic virus, or a virus thattargets particular cells, e.g., immune cells. In some embodiments, theinfectious agent targets a tumor cell and/or cancer cell in the subject.In some embodiments, the infectious agent targets an immune cell or anantigen-presenting cell (APC).

In some embodiments, the infectious agent is an oncolytic virus.Oncolytic viruses are viruses that accumulate in tumor cells andreplicate in tumor cells. By virtue of replication in the tumor cells,and optional delivery of nucleic acids encoding variant ICOSLpolypeptides or immunomodulatory polypeptides described herein, tumorcells are lysed, and the tumor shrinks and can be eliminated. Oncolyticviruses can also have a broad host and cell type range. For example,oncolytic viruses can accumulate in immunoprivileged cells orimmunoprivileged tissues, including tumors and/or metastases, and alsoincluding wounded tissues and cells, thus allowing the delivery andexpression of nucleic acids encoding the variant immunomodulatorypolypeptides described herein in a broad range of cell types. Oncolyticviruses can also replicate in a tumor cell specific manner, resulting intumor cell lysis and efficient tumor regression.

Exemplary oncolytic viruses include adenoviruses, adeno-associatedviruses, herpes viruses, Herpes Simplex Virus, Vesticular Stomaticvirus, Reovirus, Newcastle Disease virus, parvovirus, measles virus,vesticular stomatitis virus (VSV), Coxsackie virus and Vaccinia virus.In some embodiments, oncolytic viruses can specifically colonize solidtumors, while not infecting other organs, and can be used as aninfectious agent to deliver the nucleic acids encoding the variantimmunomodulatory polypeptides described herein to such solid tumors.

Oncolytic viruses for use in delivering the nucleic acids encodingvariant ICOSL polypeptides or immunomodulatory polypeptides describedherein, can be any of those known to one of skill in the art andinclude, for example, vesicular stomatitis virus, see, e.g., U.S. Pat.Nos. 7,731,974, 7,153,510, 6,653,103 and U.S. Pat. Pub. Nos.2010/0178684, 2010/0172877, 2010/0113567, 2007/0098743, 20050260601,20050220818 and EP Pat. Nos. 1385466, 1606411 and 1520175; herpessimplex virus, see, e.g., U.S. Pat. Nos. 7,897,146, 7,731,952,7,550,296, 7,537,924, 6,723,316, 6,428,968 and U.S. Pat. Pub. Nos.,2014/0154216, 2011/0177032, 2011/0158948, 2010/0092515, 2009/0274728,2009/0285860, 2009/0215147, 2009/0010889, 2007/0110720, 2006/0039894,2004/0009604, 2004/0063094, International Patent Pub. Nos., WO2007/052029, WO 1999/038955; retroviruses, see, e.g., U.S. Pat. Nos.6,689,871, 6,635,472, 5,851,529, 5,716,826, 5,716,613 and U.S. Pat. Pub.No. 20110212530; vaccinia viruses, see, e.g., 2016/0339066, andadeno-associated viruses, see, e.g., U.S. Pat. Nos. 8,007,780,7,968,340, 7,943,374, 7,906,111, 7,927,585, 7,811,814, 7,662,627,7,241,447, 7,238,526, 7,172,893, 7,033,826, 7,001,765, 6,897,045, and6,632,670.

Oncolytic viruses also include viruses that have been geneticallyaltered to attenuate their virulence, to improve their safety profile,enhance their tumor specificity, and they have also been equipped withadditional genes, for example cytotoxins, cytokines, prodrug convertingenzymes to improve the overall efficacy of the viruses (see, e.g., Kimet al., (2009) Nat Rev Cancer 9:64-71; Garcia-Aragoncillo et al., (2010)Curr Opin Mol Ther 12:403-411; see U.S. Pat. Nos. 7,588,767, 7,588,771,7,662,398 and 7,754,221 and U.S. Pat. Publ. Nos. 2007/0202572,2007/0212727, 2010/0062016, 2009/0098529, 2009/0053244, 2009/0155287,2009/0117034, 2010/0233078, 2009/0162288, 2010/0196325, 2009/0136917 and2011/0064650). In some embodiments, the oncolytic viruses can be thosethat have been modified so that they selectively replicate in cancerouscells, and, thus, are oncolytic. For example, the oncolytic virus is anadenovirus that has been engineered to have modified tropism for tumortherapy and also as gene therapy vectors. Exemplary of such is ONYX-015,H101 and Ad5ΔCR (Hallden and Portella (2012) Expert Opin Ther Targets,16:945-58) and TNFerade (McLoughlin et al. (2005) Ann. Surg. Oncol.,12:825-30), or a conditionally replicative adenovirus Oncorine®.

In some embodiments, the infectious agent is a modified herpes simplexvirus. In some embodiments, the infectious agent is a modified versionof Talimogene laherparepvec (also known as T-Vec, Imlygic or OncoVexGM-CSF), that is modified to contain nucleic acids encoding any of thevariant ICOSL polypeptides or immunomodulatory polypeptides describedherein. In some embodiments, the infectious agent is a modified herpessimplex virus that is described, e.g., in WO 2007/052029, WO1999/038955, US 2004/0063094, US 2014/0154216, or, variants thereof.

In some embodiments, the infectious agent is a virus that targets aparticular type of cells in a subject that is administered the virus,e.g., a virus that targets immune cells or antigen-presenting cells(APCs). Dendritic cells (DCs) are essential APCs for the initiation andcontrol of immune responses. DCs can capture and process antigens,migrate from the periphery to a lymphoid organ, and present the antigensto resting T cells in a major histocompatibility complex(MHC)-restricted fashion. In some embodiments, the infectious agent is avirus that specifically can target DCs to deliver nucleic acids encodingthe variant ICOSL polypeptide or immunomodulatory polypeptides forexpression in DCs. In some embodiments, the virus is a lentivirus or avariant or derivative thereof, such as an integration-deficientlentiviral vector . In some embodiments, the virus is a lentivirus thatis pseudotyped to efficiently bind to and productively infect cellsexpressing the cell surface marker dendritic cell-specific intercellularadhesion molecule-3-grabbing non-integrin (DC-SIGN), such as DCs. Insome embodiments, the virus is a lentivirus pseudotyped with a Sindbisvirus E2 glycoprotein or modified form thereof, such as those describedin WO 2013/149167. In some embodiments, the virus allows for deliveryand expression of a sequence of interest (e.g., a nucleic acid encodingany of the variant ICOSL polypeptides or immunomodulatory polypeptidesdescribed herein) to a DC. In some embodiments, the virus includes thosedescribed in WO 2008/011636, US 2011/0064763, Tareen et al. (2014) Mol.Ther., 22:575-587, or variants thereof. Exemplary of a dendriticcell-tropic vector platform is ZVex™ 2. Bacteria

In some embodiments, the infectious agent is a bacterium. For example,in some embodiments, the bacteria can deliver nucleic acids encoding anyof the variant immunomodulatory polypeptides described herein, e.g.,variant ICOSL polypeptide or immunomodulatory polypeptide, to a targetcell in the subject, such as a tumor cell, an immune cell, anantigen-presenting cell and/or a phagocytic cell. In some embodiments,the bacterium can be preferentially targeted to a specific environmentwithin a subject, such as a tumor microenvironment (TME), for expressionand/or secretion of the variant immunomodulatory polypeptides and/or totarget specific cells in the environment for expression of the variantimmunomodulatory polypeptides.

In some embodiments, the bacterium delivers the nucleic acids to thecells via bacterial-mediated transfer of plasmid DNA to mammalian cells(also referred to as “bactofection”). For example, in some embodiments,delivery of genetic material is achieved through entry of the entirebacterium into target cells. In some embodiments, spontaneous or inducedbacterial lysis can lead to the release of plasmid for subsequenteukaryotic cell expression. In some embodiments, the bacterium candeliver nucleic acids to non-phagocytic mammalian cells (e.g., tumorcells) and/or to phagocytic cells, e.g., certain immune cells and/orAPCs. In some embodiments, the nucleic acids delivered by the bacteriumcan be transferred to the nucleus of the cell in the subject forexpression. In some embodiments, the nucleic acids also includeappropriate nucleic acid sequences necessary for the expression of theoperably linked sequences encoding the variant immunomodulatorypolypeptides in a particular host cell, e.g., regulatory elements suchas promoters or enhancers. In some embodiments, the infectious agentthat is a bacterium can deliver nucleic acids encoding theimmunomodulatory proteins in the form of an RNA, such as a pre-madetranslation-competent RNA delivered to the cytoplasm of the target cellfor translation by the target cell's machinery.

In some embodiments, the bacterium can replicate and lyse the targetcells, e.g., tumor cells. In some embodiments, the bacterium can containand/or release nucleic acid sequences and/or gene products in thecytoplasm of the target cells, thereby killing the target cell, e.g.,tumor cell. In some embodiments, the infectious agent is bacterium thatcan replicate specifically in a particular environment in the subject,e.g., tumor microenvironment (TME). For example, in some embodiments,the bacterium can replicate specifically in anaerobic or hypoxicmicroenvironments. In some embodiments, conditions or factors present inparticular environments, e.g., aspartate, serine, citrate, ribose orgalactose produced by cells in the TME, can act as chemoattractants toattract the bacterium to the environment. In some embodiments, thebacterium can express and/or secrete the immunomodulatory proteinsdescribed herein in the environment, e.g., TME.

In some embodiments, the infectious agent is a bacterium that is aListeria sp., a Bifidobacterium sp., an Escherichia sp., a Closteridiumsp., a Salmonella sp., a Shigella sp., a Vibrio sp. or a Yersinia sp. Insome embodiments, the bacterium is selected from among one or more ofListeria monocytogenes, Salmonella typhimurium, Salmonella choleraesuis,Escherichia coli, Vibrio cholera, Clostridium perfringens, Clostridiumbutyricum, Clostridium novyi, Clostridium acetobutylicum,Bifidobacterium infantis, Bifidobacterium longum and Bifidobacteriumadolescentis. In some embodiments, the bacterium is an engineeredbacterium. In some embodiments, the bacterium is an engineered bacteriumsuch as those described in, e.g., Seow and Wood (2009) Molecular Therapy17(5):767-777; Baban et al. (2010) Bioengineered Bugs 1:6, 385-394;Patyar et al. (2010) J Biomed Sci 17:21; Tangney et al. (2010)Bioengineered Bugs 1:4, 284-287; van Pijkeren et al. (2010) Hum GeneTher. 21(4):405-416; WO 2012/149364; WO 2014/198002; U.S. Pat. No.9,103,831; U.S. Pat.No. 9,453,227; US 2014/0186401; US 2004/0146488; US2011/0293705; US 2015/0359909 and EP 3020816. The bacterium can bemodified to deliver nucleic acid sequences encoding any of the variantimmunomodulatory polypeptides, conjugates and/or fusions providedherein, and/or to express such variant immunomodulatory polypeptides inthe subject.

F. Nucleic acids, Vectors and Methods for Producing the Polypeptides orCells

Provided herein are isolated or recombinant nucleic acids collectivelyreferred to as “nucleic acids” which encode any of the various providedembodiments of the variant ICOSL polypeptides or immunomodulatorypolypeptides provided herein. In some embodiments, nucleic acidsprovided herein, including all described below, are useful inrecombinant production (e.g., expression) of variant ICOSL polypeptidesor immunomodulatory polypeptides provided herein. In some embodiments,nucleic acids provided herein, including all described below, are usefulin expression of variant ICOSL polypeptides or immunomodulatorypolypeptides provided herein in cells, such as in engineered cells, e.g.immune cells, or infectious agent cells. The nucleic acids providedherein can be in the form of RNA or in the form of DNA, and includemRNA, cRNA, recombinant or synthetic RNA and DNA, and cDNA. The nucleicacids provided herein are typically DNA molecules, and usuallydouble-stranded DNA molecules. However, single-stranded DNA,single-stranded RNA, double-stranded RNA, and hybrid DNA/RNA nucleicacids or combinations thereof comprising any of the nucleotide sequencesof the invention also are provided.

Also provided herein are recombinant expression vectors and recombinanthost cells useful in producing the variant ICOSL polypeptides orimmunomodulatory polypeptides provided herein.

Also provided herein are engineered cells, such as engineered immunecells, containing any of the provided nucleic acid molecules or any ofthe variant ICOSL polypeptides or immunomodulatory polypeptides, such asany of the transmembrane immunomodulatory polypeptides or secretableimmunomodulatory polypeptides.

Also provided herein are infectious agents, such as bacterial or viralcells, containing any of the provided nucleic acid molecules or any ofthe variant ICOSL polypeptides or immunomodulatory polypeptides, such asany of the transmembrane immunomodulatory polypeptides or secretableimmunomodulatory polypeptides.

In any of the above provided embodiments, the nucleic acids encoding thevariant polypeptides or immunomodulatory polypeptides provided hereincan be introduced into cells using recombinant DNA and cloningtechniques. To do so, a recombinant DNA molecule encoding aimmunomodulatory polypeptide is prepared. Methods of preparing such DNAmolecules are well known in the art. For instance, sequences coding forthe peptides could be excised from DNA using suitable restrictionenzymes. Alternatively, the DNA molecule could be synthesized usingchemical synthesis techniques, such as the phosphoramidite method. Also,a combination of these techniques could be used. In some instances, arecombinant or synthetic nucleic acid may be generated throughpolymerase chain reaction (PCR). In some embodiments, a DNA insert canbe generated encoding one or more variant ICOSL polypeptides containingat least one affinity-modified IgSF domain and, in some embodiments, asignal peptide, a transmembrane domain and/or an endodomain in accordwith the provided description. This DNA insert can be cloned into anappropriate transduction/transfection vector as is known to those ofskill in the art. Also provided are expression vectors containing thenucleic acid molecules.

In some embodiments, the expression vectors are capable of expressingthe immunomodulatory proteins in an appropriate cell under conditionssuited to expression of the protein. In some aspects, nucleic acidmolecule or an expression vector comprises the DNA molecule that encodesthe immunomodulatory protein operatively linked to appropriateexpression control sequences. Methods of effecting this operativelinking, either before or after the DNA molecule is inserted into thevector, are well known. Expression control sequences include promoters,activators, enhancers, operators, ribosomal binding sites, startsignals, stop signals, cap signals, polyadenylation signals, and othersignals involved with the control of transcription or translation.

In some embodiments, expression of the immunomodulatory protein iscontrolled by a promoter or enhancer to control or regulate expression.The promoter is operably linked to the portion of the nucleic acidmolecule encoding the variant polypeptide or immunomodulatory protein.In some embodiments, the promotor is a constitutively active promotor(such as a tissue-specific constitutively active promotor or otherconstitutive promotor). In some embodiments, the promotor is aninducible promotor, which may be responsive to an inducing agent (suchas a T cell activation signal).

In some embodiments, a constitutive promoter is operatively linked tothe nucleic acid molecule encoding the variant polypeptide orimmunomodulatory protein. Exemplary constitutive promoters include theSimian vacuolating virus 40 (SV40) promoter, the cytomegalovirus (CMV)promoter, the ubiquitin C (UbC) promoter, and the EF-1 alpha (EF1a)promoter. In some embodiments, the constitutive promoter is tissuespecific. For example, in some embodiments, the promoter allows forconstitutive expression of the immunomodulatory protein in specifictissues, such as immune cells, lymphocytes, or T cells. Exemplarytissue-specific promoters are described in U.S. Pat. No. 5,998,205,including, for example, a fetoprotein, DF3, tyrosinase, CEA, surfactantprotein, and ErbB2 promoters.

In some embodiments, an inducible promoter is operatively linked to thenucleic acid molecule encoding the variant polypeptide orimmunomodulatory protein such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription. For example, the promoter can be a regulatedpromoter and transcription factor expression system, such as thepublished tetracycline-regulated systems or other regulatable systems(see, e.g. published International PCT Appl. No. WO 01/30843), to allowregulated expression of the encoded polypeptide. An exemplaryregulatable promoter system is the Tet-On (and Tet-Off) systemavailable, for example, from Clontech (Palo Alto, Calif.). This promotersystem allows the regulated expression of the transgene controlled bytetracycline or tetracycline derivatives, such as doxycycline. Otherregulatable promoter systems are known (see e.g., published U.S.Application No. 2002-0168714, entitled “Regulation of Gene ExpressionUsing Single-Chain, Monomeric, Ligand Dependent Polypeptide Switches,”which describes gene switches that contain ligand binding domains andtranscriptional regulating domains, such as those from hormonereceptors).

In some embodiments, the promotor is responsive to an element responsiveto T-cell activation signaling. Solely by way of example, in someembodiments, an engineered T cell comprises an expression vectorencoding the immunomodulatory protein and a promotor operatively linkedto control expression of the immunomodulatory protein. The engineered Tcell can be activated, for example by signaling through an engineered Tcell receptor (TCR) or a chimeric antigen rector (CAR), and therebytriggering expression and secretion of the immunomodulatory proteinthrough the responsive promotor.

In some embodiments, an inducible promoter is operatively linked to thenucleic acid molecule encoding the immunomodulatory protein such thatthe immunomodulatory protein is expressed in response to a nuclearfactor of activated T-cells (NFAT) or nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB). For example, in some embodiments,the inducible promoter comprises a binding site for NFAT or NF-κB. Forexample, in some embodiments, the promoter is an NFAT or NF-κB promoteror a functional variant thereof. Thus, in some embodiments, the nucleicacids make it possible to control the expression of immunomodulatoryprotein while also reducing or eliminating the toxicity of theimmunomodulatory protein. In particular, engineered immune cellscomprising the nucleic acids of the invention express and secrete theimmunomodulatory protein only when the cell (e.g., a T-cell receptor(TCR) or a chimeric antigen receptor (CAR) expressed by the cell) isspecifically stimulated by an antigen and/or the cell (e.g., the calciumsignaling pathway of the cell) is non-specifically stimulated by, e.g.,phorbol myristate acetate (PMA)/Ionomycin. Accordingly, the expressionand, in some cases, secretion, of immunomodulatory protein can becontrolled to occur only when and where it is needed (e.g., in thepresence of an infectious disease-causing agent, cancer, or at a tumorsite), which can decrease or avoid undesired immunomodulatory proteininteractions.

In some embodiments, the nucleic acid encoding an immunomodulatoryprotein described herein comprises a suitable nucleotide sequence thatencodes a NFAT promoter, NF-κB promoter, or a functional variantthereof. “NFAT promoter” as used herein means one or more NFATresponsive elements linked to a minimal promoter. “NF-κB promoter”refers to one or more NF-κB responsive elements linked to a minimalpromoter. In some embodiments, the minimal promoter of a gene is aminimal human IL-2 promoter or a CMV promoter. The NFAT responsiveelements may comprise, e.g., NFAT1, NFAT2, NFAT3, and/or NFAT4responsive elements. The NFAT promoter, NF-κB promoter, or a functionalvariant thereof may comprise any number of binding motifs, e.g., atleast two, at least three, at least four, at least five, or at leastsix, at least seven, at least eight, at least nine, at least ten, atleast eleven, or up to twelve binding motifs.

The resulting recombinant expression vector having the DNA moleculethereon is used to transform an appropriate host. This transformationcan be performed using methods well known in the art. In someembodiments, a nucleic acid provided herein further comprises nucleotidesequence that encodes a secretory or signal peptide operably linked tothe nucleic acid encoding an immunomodulatory polypeptide such that aresultant soluble immunomodulatory polypeptide is recovered from theculture medium, host cell, or host cell periplasm. In other embodiments,the appropriate expression control signals are chosen to allow formembrane expression of an immunomodulatory polypeptide. Furthermore,commercially available kits as well as contract manufacturing companiescan also be utilized to make engineered cells or recombinant host cellsprovided herein.

In some embodiments, the resulting expression vector having the DNAmolecule thereon is used to transform, such as transduce, an appropriatecell. The introduction can be performed using methods well known in theart. Exemplary methods include those for transfer of nucleic acidsencoding the receptors, including via viral, e.g., retroviral orlentiviral, transduction, transposons, and electroporation. In someembodiments, the expression vector is a viral vector. In someembodiments, the nucleic acid is transferred into cells by lentiviral orretroviral transduction methods.

Any of a large number of publicly available and well-known mammalianhost cells, including mammalian T-cells or APCs, can be used in thepreparing the polypeptides or engineered cells. The selection of a cellis dependent upon a number of factors recognized by the art. Theseinclude, for example, compatibility with the chosen expression vector,toxicity of the peptides encoded by the DNA molecule, rate oftransformation, ease of recovery of the peptides, expressioncharacteristics, bio-safety and costs. A balance of these factors mustbe struck with the understanding that not all cells can be equallyeffective for the expression of a particular DNA sequence.

In some embodiments, the host cells can be a variety of eukaryoticcells, such as in yeast cells, or with mammalian cells such as Chinesehamster ovary (CHO) or HEK293 cells. Host cells can also be prokaryoticcells, such as with E. coli. The transformed recombinant host iscultured under polypeptide expressing conditions, and then purified toobtain a soluble protein. Recombinant host cells can be cultured underconventional fermentation conditions so that the desired polypeptidesare expressed. Such fermentation conditions are well known in the art.Finally, the polypeptides provided herein can be recovered and purifiedfrom recombinant cell cultures by any of a number of methods well knownin the art, including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulo sechromatography, hydrophobic interaction chromatography, and affinitychromatography. Protein refolding steps can be used, as desired, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed in the finalpurification steps.

In some embodiments, the cell is an immune cell, such as any describedabove in connection with preparing engineered cells. In someembodiments, such engineered cells are primary cells. In someembodiments, the engineered cells are autologous to the subject. In someembodiment, the engineered cells are allogeneic to the subject. In someembodiments, the engineered cells are obtained from a subject, such asby leukapheresis, and transformed ex vivo for expression of theimmunomodulatory polypeptide, e.g. transmembrane immunomodulatorypolypeptide or secretable immunomodulatory polypeptide.

Also provided are nucleic acids encoding any of the variantimmunomodulatory polypeptides contained in infectious agents describedherein. In some embodiments, the infectious agents deliver the nucleicacids to a cell in the subject, and/or permit expression of the encodedvariant polypeptides in the cell. Also provided are nucleic acids thatare used to generate, produce or modify such infectious agents. Forexample, in some embodiments, provided are vectors and/or plasmids thatcontain nucleic acids encoding the variant immunomodulatorypolypeptides, for generation of the infectious agents, delivery to thecells in a subject and/or expression of the variant immunomodulatorypolypeptides in the cells in the subject.

In some embodiments, the provided nucleic acids are recombinant viral orbacterial vectors containing nucleic acid sequences encoding the variantimmunomodulatory polypeptides. In some embodiments, the recombinantvectors can be used to produce an infectious agent that contains nucleicacid sequences encoding the variant immunomodulatory polypeptides and/orto be delivered to a target cell in the subject for expression by thetarget cell. In some embodiments, the recombinant vector is anexpression vector. In some embodiments, the recombinant vector includesappropriate sequences necessary for generation and/or production of theinfectious agent and expression in the target cell.

In some embodiments, the recombinant vector is a plasmid or cosmid.Plasmid or cosmid containing nucleic acid sequencess encoding thevariant immunomodulatory polypeptides, as described herein, is readilyconstructed using standard techniques well known in the art. Forgeneration of the infectious agent, the vector or genome can beconstructed in a plasmid form that can then be transfected into apackaging or producer cell line or a host bacterium. The recombinantvectors can be generated using any of the recombinant techniques knownin the art. In some embodiments, the vectors can include a prokaryoticorigin of replication and/or a gene whose expression confers adetectable or selectable marker such as a drug resistance forpropagation and/or selection in prokaryotic systems.

In some embodiments, the recombinant vector is a viral vector. Exemplaryrecombinant viral vectors include a lentiviral vector genome, poxvirusvector genome, vaccinia virus vector genome, adenovirus vector genome,adenovirus-associated virus vector genome, herpes virus vector genome,and alpha virus vector genome. Viral vectors can be live, attenuated,replication conditional or replication deficient, non-pathogenic(defective), replication competent viral vector, and/or is modified toexpress a heterologous gene product, e.g., the variant immunomodulatorypolypeptides provided herein. Vectors for generation of viruses also canbe modified to alter attenuation of the virus, which includes any methodof increasing or decreasing the transcriptional or translational load.

Exemplary viral vectors that can be used include modified vaccinia virusvectors (see, e.g., Guerra et al., J. Virol. 80:985-98 (2006); Tartagliaet al., AIDS Research and Human Retroviruses 8: 1445-47 (1992); Gheradiet al., J. Gen. Virol. 86:2925-36 (2005); Mayr et al., Infection 3:6-14(1975); Hu et al., J. Virol. 75: 10300-308 (2001); U.S. Pat. Nos.5,698,530, 6,998,252, 5,443,964, 7,247,615 and 7,368,116); adenovirusvector or adenovirus-associated virus vectors (see., e.g., Molin et al.,J. Virol. 72:8358-61 (1998); Narumi et al., Am J. Respir. Cell Mol.Biol. 19:936-41 (1998); Mercier et al., Proc. Natl. Acad. Sci. USA101:6188-93 (2004); U.S. Pat. Nos. 6,143,290; 6,596,535; 6,855,317;6,936,257; 7,125,717; 7,378,087; 7,550,296); retroviral vectorsincluding those based upon murine leukemia virus (MuLV), gibbon apeleukemia virus (GaLV), ecotropic retroviruses, simian immunodeficiencyvirus (SIV), human immunodeficiency virus (HIV), and combinations (see,e.g., Buchscher et al., J. Virol. 66:2731-39 (1992); Johann et al., J.Virol. 66: 1635-40 (1992); Sommerfelt et al., Virology 176:58-59 (1990);Wilson et al., J. Virol. 63:2374-78 (1989) ; Miller et al., J. Virol.65:2220-24 (1991); Miller et al., Mol. Cell Biol. 10:4239 (1990) ;Kolberg, NIH Res. 4:43 1992; Cornetta et al., Hum. Gene Ther. 2:215(1991)); lentiviral vectors including those based upon HumanImmunodeficiency Virus (HIV-1), HIV-2, feline immunodeficiency virus(FIV), equine infectious anemia virus, Simian Immunodeficiency Virus(SIV), and maedi/visna virus (see, e.g., Pfeifer et al., Annu. Rev.Genomics Hum. Genet. 2: 177-211 (2001); Zufferey et al., J. Virol. 72:9873, 1998; Miyoshi et al., J. Virol. 72:8150, 1998; Philpott andThrasher, Human Gene Therapy 18:483, 2007; Engelman et al., J. Virol.69: 2729, 1995; Nightingale et al., Mol. Therapy, 13: 1121, 2006; Brownet al., J. Virol. 73:9011 (1999); WO 2009/076524; WO 2012/141984; WO2016/011083; McWilliams et al., J. Virol. 77: 11150, 2003; Powell etal., J. Virol. 70:5288, 1996) or any, variants thereof, and/or vectorsthat can be used to generate any of the viruses described above. In someembodiments, the recombinant vector can include regulatory sequences,such as promoter or enhancer sequences, that can regulate the expressionof the viral genome, such as in the case for RNA viruses, in thepackaging cell line (see, e.g., U.S. Pat. Nos.5,385,839 and 5,168,062).

In some embodiments, the recombinant vector is an expression vector,e.g., an expression vector that permits expression of the encoded geneproduct when delivered into the target cell, e.g., a cell in thesubject, e.g., a tumor cell, an immune cell and/or an APC. In someembodiments, the recombinant expression vectors contained in theinfectious agent are capable of expressing the immunomodulatory proteinsin the target cell in the subject, under conditions suited to expressionof the protein.

In some aspects, nucleic acids or an expression vector comprises anucleic acid sequence that encodes the immunomodulatory proteinoperatively linked to appropriate expression control sequences. Methodsof affecting this operative linking, either before or after the nucleicacid sequence encoding the immunomodulatory protein is inserted into thevector, are well known. Expression control sequences include promoters,activators, enhancers, operators, ribosomal binding sites, startsignals, stop signals, cap signals, polyadenylation signals, and othersignals involved with the control of transcription or translation. Thepromoter can be operably linked to the portion of the nucleic acidsequence encoding the immunomodulatory protein. In some embodiments, thepromotor is a constitutively active promotor in the target cell (such asa tissue-specific constitutively active promotor or other constitutivepromotor). For example, the recombinant expression vector may alsoinclude, lymphoid tissue-specific transcriptional regulatory elements(TRE) such as a B lymphocyte, T lymphocyte, or dendritic cell specificTRE. Lymphoid tissue specific TRE are known in the art (see, e.g.,Thompson et al., Mol. Cell. Biol. 12:1043-53 (1992); Todd et al., J.Exp. Med. 177:1663-74 (1993); Penix et al., J. Exp. Med. 178:1483-96(1993)). In some embodiments, the promotor is an inducible promotor,which may be responsive to an inducing agent (such as a T cellactivation signal). In some embodiments, nucleic acids delivered to thetarget cell in the subject, e.g., tumor cell, immune cell and/or APC,can be operably linked to any of the regulatory elements describedabove.

In some embodiments, the vector is a bacterial vector, e.g., a bacterialplasmid or cosmid. In some embodiments, the bacterial vector isdelivered to the target cell, e.g., tumor cells, immune cells and/orAPCs, via bacterial-mediated transfer of plasmid DNA to mammalian cells(also referred to as “bactofection”). In some embodiments, the deliveredbacterial vector also contains appropriate expression control sequencesfor expression in the target cells, such as a promoter sequence and/orenhancer sequences, or any regulatory or control sequences describedabove. In some embodiments, the bacterial vector contains appropriateexpression control sequences for expression and/or secretion of theencoded variant polypeptides in the infectious agent, e.g., thebacterium.

In some embodiments, polypeptides provided herein can also be made bysynthetic methods. Solid phase synthesis is the preferred technique ofmaking individual peptides since it is the most cost-effective method ofmaking small peptides. For example, well known solid phase synthesistechniques include the use of protecting groups, linkers, and solidphase supports, as well as specific protection and deprotection reactionconditions, linker cleavage conditions, use of scavengers, and otheraspects of solid phase peptide synthesis. Peptides can then be assembledinto the polypeptides as provided herein.

IV. METHODS OF ASSESSING ACTIVITY IMMUNE MODULATION OF VARIANT ICOSLPOLYPEPTIDES AND IMMUNOMODULATORY PROTEINS

In some embodiments, the variant ICOSL polypeptides provided herein(e.g. full-length and/or specific binding fragments or conjugates, stackconstructs or fusion thereof) exhibit immunomodulatory activity tomodulate T cell activation. In some embodiments, ICOSL polypeptidesmodulate IFN-gamma expression in a primary T cell assay relative to awild-type or unmodified ICOSL control. In some cases, modulation ofIFN-gamma expression can increase or decrease IFN-gamma expressionrelative to the control. Assays to determine specific binding andIFN-gamma expression are well-known in the art and include the MLR(mixed lymphocyte reaction) assays measuring interferon-gamma cytokinelevels in culture supernatants (Wang et al., Cancer Immunol Res. 2014September: 2(9):846-56), SEB (staphylococcal enterotoxin B) T cellstimulation assay (Wang et al., Cancer Immunol Res. 2014 September:2(9):846-56), and anti-CD3 T cell stimulation assays (Li and Kurlander,J Transl Med. 2010: 8: 104).

In some embodiments, a variant ICOSL polypeptide can in some embodimentsincrease or, in alternative embodiments, decrease IFN-gamma(interferon-gamma) expression in a primary T-cell assay relative to awild-type ICOSL control. In some embodiments of the providedpolypeptides containing a soluble variant ICOSL sequence, thepolypeptide can increase IFN-gamma expression and, in alternativeembodiments, decrease IFN-gamma expression in a primary T-cell assayrelative to a wild-type ICOSL control. In some embodiments of theprovided polypeptides containing multiple variant ICOSL sequences, thepolypeptide can increase IFN-gamma expression and, in alternativeembodiments, decrease IFN-gamma expression in a primary T-cell assayrelative to a wild-type ICOSL control.

Those of skill will recognize that the format of the primary T-cellassay used to determine an increase in IFN-gamma expression can differfrom that employed to assay for a decrease in IFN-gamma expression. Inassaying for the ability of a variant ICOSL to decrease IFN-gammaexpression in a primary T-cell assay, a Mixed Lymphocyte Reaction (MLR)assay can be used as described in Example 6. In some cases, a solubleform of a variant ICOSL can be employed to determine the ability of thevariant ICOSL to antagonize and thereby decrease the IFN-gammaexpression in a MLR as likewise described in Example 6.

Alternatively, in assaying for the ability of a variant ICOSL toincrease IFN-gamma expression in a primary T-cell assay, aco-immobilization assay can be used as described in Example 6. In aco-immobilization assay, a TCR signal, provided in some embodiments byanti-CD3 antibody, is used in conjunction with a co-immobilized variantICOSL to determine the ability to increase IFN-gamma expression relativeto an ICOSL control. In some cases, a soluble form of a variant ICOSLthat is multimerized to a degree to provide multivalent binding can beemployed to determine the ability of the variant ICOSL to agonize andthereby increase the IFN-gamma expression in a MLR as likewise describedin Example 6.

Use of proper controls is known to those of skill in the art, however,in the aforementioned embodiments, the control typically involves use ofthe unmodified ICOSL, such as a wild-type of native ICOSL isoform fromthe same mammalian species from which the variant ICOSL was derived ordeveloped. Irrespective of whether the binding affinity to either one orboth of ICOS and CD28 is increased or decreased, a variant ICOSL in someembodiments will increase IFN-gamma expression and, in alternativeembodiments, decrease IFN-gamma expression in a primary T-cell assayrelative to a wild-type ICOSL control.

In some embodiments, a variant ICOSL increases IFN-gamma expression(i.e., protein expression) relative to a wild-type or unmodified ICOSLcontrol by at least: 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, orhigher. In other embodiments, a variant ICOSL decreases IFN-gammaexpression (i.e. protein expression) relative to a wild-type orunmodified ICOSL control by at least: 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or higher. In some embodiments, the wild-type ICOSLcontrol is murine ICOSL, such as would typically be used for a variantICOSL altered in sequence from that of a wild-type murine ICOSLsequence. In some embodiments, the wild-type ICOSL control is humanICOSL, such as would typically be used for a variant ICOSL altered insequence from that of a wild-type human ICOSL sequence such as an ICOSLsequence comprising the sequence of amino acids of SEQ ID NO:32 or SEQID NO:196.

V. PHARMACEUTICAL FORMULATIONS

Provided herein are compositions containing any of the variant ICOSLpolypeptides, immomodulatory proteins, conjugates, engineered cells orinfectious agents described herein. The pharmaceutical composition canfurther comprise a pharmaceutically acceptable exicipient. For example,the pharmaceutical composition can contain one or more excipients formodifying, maintaining or preserving, for example, the pH, osmolarity,viscosity, clarity, color, isotonicity, odor, sterility, stability, rateof dissolution or release, adsorption, or penetration of thecomposition. In some aspects, a skilled artisan understands that apharmaceutical composition containing cells may differ from apharmaceutical composition containing a protein.

In some embodiments, the pharmaceutical composition is a solid, such asa powder, capsule, or tablet. For example, the components of thepharmaceutical composition can be lyophilized. In some embodiments, thesolid pharmaceutical composition is reconstituted or dissolved in aliquid prior to administration.

In some embodiments, the pharmaceutical composition is a liquid, forexample variant ICOSL polypeptides dissolved in an aqueous solution(such as physiological saline or Ringer's solution). In someembodiments, the pH of the pharmaceutical composition is between about4.0 and about 8.5 (such as between about 4.0 and about 5.0, betweenabout 4.5 and about 5.5, between about 5.0 and about 6.0, between about5.5 and about 6.5, between about 6.0 and about 7.0, between about 6.5and about 7.5, between about 7.0 and about 8.0, or between about 7.5 andabout 8.5).

In some embodiments, the pharmaceutical composition comprises apharmaceutically-acceptable excipient, for example a filler, binder,coating, preservative, lubricant, flavoring agent, sweetening agent,coloring agent, a solvent, a buffering agent, a chelating agent, orstabilizer. Examples of pharmaceutically-acceptable fillers includecellulose, dibasic calcium phosphate, calcium carbonate,microcrystalline cellulose, sucrose, lactose, glucose, mannitol,sorbitol, maltol, pregelatinized starch, corn starch, or potato starch.Examples of pharmaceutically-acceptable binders includepolyvinylpyrrolidone, starch, lactose, xylitol, sorbitol, maltitol,gelatin, sucrose, polyethylene glycol, methyl cellulose, or cellulose.Examples of pharmaceutically-acceptable coatings include hydroxypropylmethylcellulose (HPMC), shellac, corn protein zein, or gelatin. Examplesof pharmaceutically-acceptable disintegrants includepolyvinylpyrrolidone, carboxymethyl cellulose, or sodium starchglycolate. Examples of pharmaceutically-acceptable lubricants includepolyethylene glycol, magnesium stearate, or stearic acid. Examples ofpharmaceutically-acceptable preservatives include methyl parabens, ethylparabens, propyl paraben, benzoic acid, or sorbic acid. Examples ofpharmaceutically-acceptable sweetening agents include sucrose,saccharine, aspartame, or sorbitol. Examples ofpharmaceutically-acceptable buffering agents include carbonates,citrates, gluconates, acetates, phosphates, or tartrates.

In some embodiments, the pharmaceutical composition further comprises anagent for the controlled or sustained release of the product, such asinjectable microspheres, bio-erodible particles, polymeric compounds(polylactic acid, polyglycolic acid), beads, or liposomes.

In some embodiments, the pharmaceutical composition is sterile.Sterilization may be accomplished by filtration through sterilefiltration membranes or radiation. Where the composition is lyophilized,sterilization using this method may be conducted either prior to orfollowing lyophilization and reconstitution. The composition forparenteral administration may be stored in lyophilized form or insolution. In addition, parenteral compositions generally are placed intoa container having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

In some embodiments, provided are pharmaceutical compositions containingthe transmembrane immunomodulatory proteins, including engineered cellsexpressing such transmembrane immunomodulatory proteins. In someembodiments, the pharmaceutical compositions and formulations includeone or more optional pharmaceutically acceptable carrier or excipient.Such compositions may comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.Compositions of the present invention are preferably formulated forintravenous administration.

In some embodiments, the pharmaceutical composition contains infectiousagents containing nucleic acid sequences encoding the immunomodulatoryvariant polypeptides. In some embodiments, the pharmaceuticalcomposition contains a dose of infectious agents suitable foradministration to a subject that is suitable for treatment. In someembodiments, the pharmaceutical composition contains an infectious agentthat is a virus, at a single or multiple dosage amount, of between aboutbetween or between about 1×10⁵ and about 1×10¹² plaque-forming units(pfu), 1×10⁶ and 1×10¹⁰ pfu, or 1×10⁷ and 1×10¹⁰ pfu, each inclusive,such as at least or at least about or at about 1×10⁶, 1×10, 1×10⁸,1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹pfu or about 1×10¹⁰ pfu. In someembodiments, the pharmaceutical composition can contain a virusconcentration of from or from about 10⁵ to about 10¹⁰ pfu/mL, forexample, 5×10⁶ to 5×10⁹ or 1×10⁷ to 1×10⁹ pfu/mL, such as at least or atleast about or at about 10⁶ pfu/mL, 10⁷ pfu/mL, 10⁸ pfu/mL or 10⁹pfu/mL. In some embodiments, the pharmaceutical composition contains aninfectious agent that is a bacterium, at a single or multiple dosageamount, of between about between or between about 1×10³ and about 1 ×10⁹colony-forming units (cfu), 1 ×10⁴ and 1 ×10⁹ cfu, or 1 ×10⁵ and 1 ×10⁷cfu, each inclusive, such as at least or at least about or at about1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸ or 1×10⁹ cfu. In some embodiments, thepharmaceutical composition can contain a bacterial concentration of fromor from about 10³ to about 10⁸ cfu/mL, for example, 5×10⁵ to 5×10⁷ or1×10⁶ to 1×10⁷ cfu/mL, such as at least or at least about or at about10⁵ cfu/mL, 10⁶ cfu/mL, 10⁷ cfu/mL or 10⁸ cfu/mL.

Such a formulation may, for example, be in a form suitable forintravenous infusion. A pharmaceutically acceptable carrier may be apharmaceutically acceptable material, composition, or vehicle that isinvolved in carrying or transporting cells of interest from one tissue,organ, or portion of the body to another tissue, organ, or portion ofthe body. For example, the carrier may be a liquid or solid filler,diluent, excipient, solvent, or encapsulating material, or somecombination thereof. Each component of the carrier must be“pharmaceutically acceptable” in that it must be compatible with theother ingredients of the formulation. It also must be suitable forcontact with any tissue, organ, or portion of the body that it mayencounter, meaning that it must not carry a risk of toxicity,irritation, allergic response, immunogenicity, or any other complicationthat excessively outweighs its therapeutic benefits.

In some embodiments, the pharmaceutical composition is administered to asubject. Generally, dosages and routes of administration of thepharmaceutical composition are determined according to the size andcondition of the subject, according to standard pharmaceutical practice.For example, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models such asmice, rats, rabbits, dogs, pigs, or monkeys. An animal model may also beused to determine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans. The exact dosage will bedetermined in light of factors related to the subject requiringtreatment. Dosage and administration are adjusted to provide sufficientlevels of the active compound or to maintain the desired effect. Factorsthat may be taken into account include the severity of the diseasestate, the general health of the subject, the age, weight, and gender ofthe subject, time and frequency of administration, drug combination(s),reaction sensitivities, and response to therapy.

Long-acting pharmaceutical compositions may be administered every 3 to 4days, every week, or biweekly depending on the half-life and clearancerate of the particular formulation. The frequency of dosing will dependupon the pharmacokinetic parameters of the molecule in the formulationused. Typically, a composition is administered until a dosage is reachedthat achieves the desired effect. The composition may therefore beadministered as a single dose, or as multiple doses (at the same ordifferent concentrations/dosages) over time, or as a continuousinfusion. Further refinement of the appropriate dosage is routinelymade. Appropriate dosages may be ascertained through use of appropriatedose-response data. A number of biomarkers or physiological markers fortherapeutic effect can be monitored including T cell activation orproliferation, cytokine synthesis or production (e.g., production ofTNF-α, IFN-γ, IL-2), induction of various activation markers (e.g.,CD25, IL-2 receptor), inflammation, joint swelling or tenderness, serumlevel of C-reactive protein, anti-collagen antibody production, and/or Tcell-dependent antibody response(s).

In some embodiments, the pharmaceutical composition is administered to asubject through any route, including orally, transdermally, byinhalation, intravenously, intra-arterially, intramuscularly, directapplication to a wound site, application to a surgical site,intraperitoneally, by suppository, subcutaneously, intradermally,transcutaneously, by nebulization, intrapleurally, intraventricularly,intra-articularly, intraocularly, or intraspinally.

In some embodiments, the dosage of the pharmaceutical composition is asingle dose or a repeated dose. In some embodiments, the doses are givento a subject once per day, twice per day, three times per day, or fouror more times per day. In some embodiments, about 1 or more (such asabout 2 or more, about 3 or more, about 4 or more, about 5 or more,about 6 or more, or about 7 or more) doses are given in a week. In someembodiments, multiple doses are given over the course of days, weeks,months, or years. In some embodiments, a course of treatment is about 1or more doses (such as about 2 or more does, about 3 or more doses,about 4 or more doses, about 5 or more doses, about 7 or more doses,about 10 or more doses, about 15 or more doses, about 25 or more doses,about 40 or more doses, about 50 or more doses, or about 100 or moredoses).

In some embodiments, an administered dose of the pharmaceuticalcomposition is about 1 μg of protein per kg subject body mass or more(such as about 2 μg of protein per kg subject body mass or more, about 5μg of protein per kg subject body mass or more, about 10 μg of proteinper kg subject body mass or more, about 25 μg of protein per kg subjectbody mass or more, about 50 μg of protein per kg subject body mass ormore, about 100 μg of protein per kg subject body mass or more, about250 μg of protein per kg subject body mass or more, about 500 μg ofprotein per kg subject body mass or more, about 1 mg of protein per kgsubject body mass or more, about 2 mg of protein per kg subject bodymass or more, or about 5 mg of protein per kg subject body mass ormore).

In some embodiments, a therapeutic amount of a cell composition isadministered. Typically, precise amount of the compositions of thepresent invention to be administered can be determined by a physicianwith consideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising engineered cells, e.g. T cells, as described herein may beadministered at a dosage of 104 to 109 cells/kg body weight, such as 105to 106 cells/kg body weight, including all integer values within thoseranges. Engineered cell compositions, such as T cell compositions, mayalso be administered multiple times at these dosages. The cells can beadministered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al, New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particularpatient can readily be determined by one skilled in the art of medicineby monitoring the patient for signs of disease and adjusting thetreatment accordingly.

A variety of means are known for determining if administration of atherapeutic composition of the invention sufficiently modulatesimmunological activity by eliminating, sequestering, or inactivatingimmune cells mediating or capable of mediating an undesired immuneresponse; inducing, generating, or turning on immune cells that mediateor are capable of mediating a protective immune response; changing thephysical or functional properties of immune cells; or a combination ofthese effects. Examples of measurements of the modulation ofimmunological activity include, but are not limited to, examination ofthe presence or absence of immune cell populations (using flowcytometry, immunohistochemistry, histology, electron microscopy,polymerase chain reaction (PCR)); measurement of the functional capacityof immune cells including ability or resistance to proliferate or dividein response to a signal (such as using T-cell proliferation assays andpepscan analysis based on 3H-thymidine incorporation followingstimulation with anti-CD3 antibody, anti-T-cell receptor antibody,anti-CD28 antibody, calcium ionophores, PMA (phorbol 12-myristate13-acetate) antigen presenting cells loaded with a peptide or proteinantigen; B cell proliferation assays); measurement of the ability tokill or lyse other cells (such as cytotoxic T cell assays); measurementsof the cytokines, chemokines, cell surface molecules, antibodies andother products of the cells (e.g., by flow cytometry, enzyme-linkedimmunosorbent assays, Western blot analysis, protein microarrayanalysis, immunoprecipitation analysis); measurement of biochemicalmarkers of activation of immune cells or signaling pathways withinimmune cells (e.g., Western blot and immunoprecipitation analysis oftyrosine, serine or threonine phosphorylation, polypeptide cleavage, andformation or dissociation of protein complexes; protein array analysis;DNA transcriptional, profiling using DNA arrays or subtractivehybridization); measurements of cell death by apoptosis, necrosis, orother mechanisms (e.g., annexin V staining, TUNEL assays, gelelectrophoresis to measure DNA laddering, histology; fluorogenic caspaseassays, Western blot analysis of caspase substrates); measurement of thegenes, proteins, and other molecules produced by immune cells (e.g.,Northern blot analysis, polymerase chain reaction, DNA microarrays,protein microarrays, 2-dimensional gel electrophoresis, Western blotanalysis, enzyme linked immunosorbent assays, flow cytometry); andmeasurement of clinical symptoms or outcomes such as improvement ofautoimmune, neurodegenerative, and other diseases involvingself-proteins or self-polypeptides (clinical scores, requirements foruse of additional therapies, functional status, imaging studies) forexample, by measuring relapse rate or disease severity (using clinicalscores known to the ordinarily skilled artisan) in the case of multiplesclerosis, measuring blood glucose in the case of type I diabetes, orjoint inflammation in the case of rheumatoid arthritis.

VI. ARTICLES OF MANUFACTURE AND KITS

Also provided herein are articles of manufacture comprising thepharmaceutical compositions described herein in suitable packaging.Suitable packaging for compositions (such as ophthalmic compositions)described herein are known in the art, and include, for example, vials(such as sealed vials), vessels, ampules, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Thesearticles of manufacture may further be sterilized and/or sealed.

Further provided are kits comprising the pharmaceutical compositions (orarticles of manufacture) described herein, which may further compriseinstruction(s) on methods of using the composition, such as usesdescribed herein. The kits described herein may also include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for performing any methods described herein.

VII. THERAPEUTIC APPLICATIONS

The pharmaceutical compositions described herein (includingpharmaceutical composition comprising the variant ICOSL polypeptides,the immunomodulatory proteins, the conjugates, the engineered cells andinfectious agents described herein) can be used in a variety oftherapeutic applications, such as the treatment of a disease. Forexample, in some embodiments the pharmaceutical composition is used totreat inflammatory or autoimmune disorders, cancer, organtransplantation, viral infections, and/or bacterial infections in amammal. The pharmaceutical composition can modulate (e.g. increase ordecrease) an immune response to treat the disease. In some embodiments,the provided methods are applicable to therapeutic administration ofvariant ICOSL polypeptides, the immunomodulatory proteins, theconjugates, the engineered cells and infectious agents described herein.It is within the level of a skilled artisan, in view of the provideddisclosure, to choose a format for the indication depending on the typeof modulation of the immune response, e.g. increase or decrease that isdesired.

In some embodiments, a pharmaceutical composition provided herein thatstimulates the immune response is administered, which can be useful, forexample, in the treatment of cancer, viral infections, or bacterialinfections. In some embodiments, the pharmaceutical composition containsa variant ICOSL polypeptide in a format that exhibits agonist activityof its cognate binding partner CD28 or ICOS and/or that stimulates orinitiates costimulatory signaling via CD28 or ICOS. Exemplary formats ofan ICOSL polypeptide for use in connection with such therapeuticapplications include, for example, an immunomodulatory protein or“stack” of a variant ICOSL polypeptide and an IgSF domain or variantthereof that binds to a tumor antigen (e.g. Nkp30 or affinity-modifiedvariant) (also called a “tumor-localizing IgSF domain), a conjugatecontaining a variant ICOSL polypeptide linked to a tumor-targetingmoiety (also called a tumor-localizing moiety), an engineered cellexpressing a transmembrane immunomodulatory protein or an infectiousagent comprising a nucleic acid molecule encoding a transmembraneimmunomodulatory protein, such as for expression of the transmembraneimmunomodulatory protein in an infected cell (e.g. tumor cell or APC,e.g. dendritic cell).

In some embodiments, the pharmaceutical composition can be used toinhibit growth of mammalian cancer cells (such as human cancer cells). Amethod of treating cancer can include administering an effective amountof any of the pharmaceutical compositions described herein to a subjectwith cancer. The effective amount of the pharmaceutical composition canbe administered to inhibit, halt, or reverse progression of cancers.

The cancers that can be treated by the pharmaceutical compositions andthe treatment methods described herein include, but are not limited to,melanoma, bladder cancer, hematological malignancies (leukemia,lymphoma, myeloma), liver cancer, brain cancer, renal cancer, breastcancer, pancreatic cancer (adenocarcinoma), colorectal cancer, lungcancer (small cell lung cancer and non-small-cell lung cancer), spleencancer, cancer of the thymus or blood cells (i.e., leukemia), prostatecancer, testicular cancer, ovarian cancer, uterine cancer, gastriccarcinoma, a musculoskeletal cancer, a head and neck cancer, agastrointestinal cancer, a germ cell cancer, or an endocrine andneuroendocrine cancer. In some embodiments, the cancer is Ewing'ssarcoma. In some embodiments, the cancer is selected a from melanoma,lung cancer, bladder cancer, and a hematological malignancy. In someembodiments, the cancer is a lymphoma, lymphoid leukemia, myeloidleukemia, cervical cancer, neuroblastoma, or multiple myeloma.

Human cancer cells can be treated in vivo, or ex vivo. In ex vivotreatment of a human patient, tissue or fluids containing cancer cellsare treated outside the body and then the tissue or fluids arereintroduced back into the patient. In some embodiments, the cancer istreated in a human patient in vivo by administration of the therapeuticcomposition into the patient.

In some embodiments, the pharmaceutical composition is administered as amonotherapy (i.e., as a single agent) or as a combination therapy (i.e.,in combination with one or more additional anticancer agents, such as achemotherapeutic drug, a cancer vaccine, or an immune checkpointinhibitor. In some embodiments, the pharmaceutical composition can alsobe administered with radiation therapy.

In some embodiments, the pharmaceutical composition suppresses an immuneresponse, which can be useful in the treatment of inflammatory orautoimmune disorders, or organ transplantation. In some embodiments, thepharmaceutical composition contains a variant ICOSL polypeptide in aformat that exhibits antagonist activity of its cognate binding partnerCD28 or ICOS and/or that blocks or inhibits costimulatory signaling viaCD28 or ICOS. Exemplary formats of an ICOSL polypeptide for use inconnection with such therapeutic applications include, for example, avariant ICOSL polypeptide that is soluble (e.g. variant ICOSL-Fc fusionprotein), an immunomodulatory protein or “stack” of a variant ICOSLpolypeptide and another IgSF domain, including soluble forms thereofthat are Fc fusions, an engineered cell expressing a secretableimmunomodulatory protein, or an infectious agent comprising a nucleicacid molecule encoding a secretable immunomodulatory protein, such asfor expression and secretion of the secretable immunomodulatory proteinin an infected cell (e.g. tumor cell or APC, e.g. dendritic cell).

In some embodiments, the inflammatory or autoimmune disorder isAntineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, avasculitis, an autoimmune skin disease, transplantation, a Rheumaticdisease, an inflammatory gastrointestinal disease, an inflammatory eyedisease, an inflammatory neurological disease, an inflammatory pulmonarydisease, an inflammatory endocrine disease, or an autoimmunehematological disease.

In some embodiments, the inflammatory and autoimmune disorders that canbe treated by the pharmaceutical composition described herein isAddison's Disease, allergies, alopecia areata, Alzheimer's,antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis,ankylosing spondylitis, antiphospholipid syndrome (Hughes Syndrome),asthma, atherosclerosis, rheumatoid arthritis, autoimmune hemolyticanemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmunelymphoproliferative syndrome, autoimmune myocarditis, autoimmuneoophoritis, autoimmune orchitis, azoospermia, Behcet's Disease, Berger'sDisease, bullous pemphigoid, cardiomyopathy, cardiovascular disease,celiac Sprue/coeliac disease, chronic fatigue immune dysfunctionsyndrome (CFIDS), chronic idiopathic polyneuritis, chronic inflammatorydemyelinating, polyradicalneuropathy (CIPD), chronic relapsingpolyneuropathy (Guillain-Barré syndrome), Churg-Strauss Syndrome (CSS),cicatricial pemphigoid, cold agglutinin disease (CAD), COPD (chronicobstructive pulmonary disease), CREST syndrome, Crohn's disease,dermatitis, herpetiformus, dermatomyositis, diabetes, discoid lupus,eczema, epidermolysis bullosa acquisita, essential mixedcryoglobulinemia, Evan's Syndrome, exopthalmos, fibromyalgia,Goodpasture's Syndrome, Graves' Disease, Hashimoto's thyroiditis,idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura(ITP), IgA nephropathy, immunoproliferative disease or disorder,inflammatory bowel disease (IBD), interstitial lung disease, juvenilearthritis, juvenile idiopathic arthritis (JIA), Kawasaki's Disease,Lambert-Eaton Myasthenic Syndrome, lichen planus, lupus nephritis,lymphoscytic lypophisitis, Ménière's Disease, Miller Fish Syndrome/acutedisseminated encephalomyeloradiculopathy, mixed connective tissuedisease, multiple sclerosis (MS), muscular rheumatism, myalgicencephalomyelitis (ME), myasthenia gravis, ocular inflammation,pemphigus foliaceus, pemphigus vulgaris, pernicious anaemia,polyarteritis nodosa, polychondritis, polyglandular syndromes(Whitaker's syndrome), polymyalgia rheumatica, polymyositis, primaryagammaglobulinemia, primary biliary cirrhosis/autoimmune cholangiopathy,psoriasis, psoriatic arthritis, Raynaud's Phenomenon, Reiter'sSyndrome/reactive arthritis, restenosis, rheumatic fever, rheumaticdisease, sarcoidosis, Schmidt's syndrome, scleroderma, Sjörgen'sSyndrome, stiff-man syndrome, systemic lupus erythematosus (SLE),systemic scleroderma, Takayasu arteritis, temporal arteritis/giant cellarteritis, thyroiditis, Type 1 diabetes, ulcerative colitis, uveitis,vasculitis, vitiligo, interstitial bowel disease or Wegener'sGranulomatosis. In some embodiments, the inflammatory or autoimmunedisorder is selected from interstitial bowel disease, transplant,Crohn's disease, ulcerative colitis, multiple sclerosis, asthma,rheumatoid arthritis, and psoriasis.

In some embodiments, the pharmaceutical composition is administered tomodulate an autoimmune condition. For example, suppressing an immuneresponse can be beneficial in methods for inhibiting rejection of atissue, cell, or organ transplant from a donor by a recipient.Accordingly, in some embodiments, the pharmaceutical compositionsdescribed herein are used to limit or prevent graft-related ortransplant related diseases or disorders, such as graft versus hostdisease (GVHD). In some embodiments, the pharmaceutical compositions areused to suppress autoimmune rejection of transplanted or grafted bonemarrow, organs, skin, muscle, neurons, islets, or parenchymal cells.

Pharmaceutical compositions comprising engineered cells and the methodsdescribed herein can be used in adoptive cell transfer applications. Insome embodiments, cell compositions comprising engineered cells can beused in associated methods to, for example, modulate immunologicalactivity in an immunotherapy approach to the treatment of, for example,a mammalian cancer or, in other embodiments the treatment of autoimmunedisorders. The methods employed generally comprise a method ofcontacting a TIP of the present invention with a mammalian cell underconditions that are permissive to specific binding of the affinitymodified IgSF domain and modulation of the immunological activity of themammalian cell. In some embodiments, immune cells (such as tumorinfiltrating lymphocytes (TILs), T-cells (including CD8+ or CD4+T-cells), or APCs) are engineered to express as a membrane proteinand/or as a soluble variant ICOSL polypeptide, immunomodulatory protein,or conjugate as described herein. The engineered cells can then contacta mammalian cell, such as an APC, a second lymphocyte or tumor cell inwhich modulation of immunological activity is desired and underconditions that are permissive of specific binding of the affinitymodified IgSF domain to a counter-structure on the mammalian cell suchthat immunological activity can be modulated in the mammalian cell.Cells can be contacted in vivo or ex vivo.

In some embodiments, the engineered cells are autologous cells. In otherembodiments, the cells are allogeneic. In some embodiments, the cellsare autologous engineered cells reinfused into the mammal from which itwas isolated. In some embodiments, the cells are allogenic engineeredcells infused into the mammal. In some embodiments, the cells areharvested from a patient's blood or tumor, engineered to express apolypeptide (such as the variant ICOSL polypeptide, immunomodulatoryprotein, or conjugate as described herein), expanded in an in vitroculture system (for example, by stimulating the cells), and reinfusedinto the patient to mediate tumor destruction. In some embodiments, themethods are conducted by adoptive cell transfer wherein cells expressingthe TIP (e.g., a T-cell) are infused back into the patient. In someembodiments, the therapeutic compositions and methods of the inventionare used in the treatment of a mammalian patient of cancers such aslymphoma, lymphoid leukemia, myeloid leukemia, cervical cancer,neuroblastoma, or multiple myeloma.

VIII. EXEMPLARY EMBODIMENTS

Among the provided embodiments are:

1. A variant ICOS Ligand (ICOSL) polypeptide, comprising an IgV domainor specific binding fragment thereof, an IgC domain or a specificbinding fragment thereof, or both, wherein the variant ICOSL polypeptidecomprises one or more amino acid modifications in an unmodified ICOSL ora specific binding fragment thereof corresponding to position(s)selected from 10, 11, 13, 16, 18, 20, 25, 27, 30, 33, 37, 38, 42, 43,47, 52, 54, 57, 61, 62, 67, 71, 72, 74, 75, 77, 78, 80, 84, 89, 90, 92,93, 94, 96, 97, 98, 99, 100, 102, 103, 107, 109, 110, 111, 113, 115,116, 117, 119, 120, 121, 122, 126, 129, 130, 132, 133, 135, 138, 139,140, 142, 143, 144, 146, 148, 151, 152, 153, 154, 155, 156, 158, 161,164, 166, 168, 172, 173, 175, 190, 192, 193, 194, 198, 201, 203, 207,208, 210, 212, 217, 218, 220, 221, 224, 225, or 227 with reference toSEQ ID NO:32.

2. The variant ICOSL polypeptide of embodiment 1, wherein the unmodifiedICOSL is a mammalian ICOSL or a specific binding fragment thereof.

3. The variant ICOSL polypeptide of embodiment 2, wherein the unmodifiedICOSL is a human ICOSL or a specific binding fragment thereof.

4. The variant ICOSL polypeptide of any one of embodiments 1-3, whereinthe unmodified ICOSL comprises (i) the sequence of amino acids set forthin SEQ ID NO:32, (ii) a sequence of amino acids that has at least 95%sequence identity to SEQ ID NO:32; or (iii) a portion thereof comprisingan IgV domain or IgC domain or specific binding fragments thereof orboth.

5. The variant ICOSL polypeptide of any one of embodiments 1-4, wherein:the specific binding fragment of the IgV domain or IgC domain has alength of at least 50, 60, 70, 80, 90, 100, 110 or more amino acids; or

the specific binding fragment of the IgV domain comprises a length thatis at least 80% of the length of the IgV domain set for as amino acids19-129 of SEQ ID NO:5 and/or the specific binding fragment of the IgCdomain comprises a length that is at least 80% of the length of the IgCdomain set forth as amino acids 141-227 of SEQ ID NO:5.

6. The variant ICOSL polypeptide of any one of embodiments 1-5, whereinthe variant ICOSL comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications, optionallyamino acid substitutions, insertions and/or deletions.

7. The variant ICOSL of any of embodiments 1-6, wherein the variantICOSL comprises a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to SEQ ID NO:32 or a specific binding fragmentthereof.

8. The variant ICOSL polypeptide of any of embodiments 1-7, wherein thevariant ICOSL polypeptide exhibits altered binding to the ectodomain ofICOS, CD28 or CTLA-4 compared to the unmodified ICOSL.

9. The variant ICOSL polypeptide of any of embodiments 1-8, wherein thevariant ICOSL polypeptide exhibits altered binding to the ectodomain ofICOS or CD28 compared to the unmodified ICOSL.

10. The variant ICOSL polypeptide of embodiment 8 or embodiment 9,wherein the altered binding is altered binding affinity and/or alteredbinding selectivity.

11. The variant ICOSL polypeptide of any of embodiments 1-10, whereinthe one or more amino acid modifications are selected from M10V, M10I,V11E, S13G, E16V, S18R, A20V, S25G, F27S, F27C, N30D, Y33del, Q37R,K42E, T43A, Y47H, N52H, N52D, N52Q, N52S, N52Y, N52K, S54A, S54P, N57D,N57Y, R61S, R61C, Y62F, L67P, A71T, G72R, L74Q, R75Q, D77G, F78L, L80P,N84Q, E90A, K92R, F93L, H94E, H94D, L96F, L961, V97A, L98F, S99G, Q100R,Q100K, Q100P, L102R, G103E, V107A, V1071, S109G, S109N, V110D, V110N,V110A, E111del, T113E, H115Q, H115R, V116A, A117T, N119Q, F1201, F120S,S121G, V122A, V122M, S126T, S126R, H129P, S130G,S132F, Q133H, E135K,F138L, T139S, C140del, S142F,I143V, I143T, N144D, Y146C, V151A, Y152C,Y152H,W153R, I154F, N155H, N155Q, K156M, D158G, L161P, L161M, L166Q,N168Q, F172S, L173S, M175T, T190A, T190S, S192G, V193M, N194D, C198R,N201S, L203P, L203F, N207Q, L208P, V210A, S212G, D217V, I218T, 1218N,E220G, R221G, R221I, I224V, T225A, N227K, or a conservative amino acidsubstitution thereof.

12. The variant ICOSL polypeptide of any of embodiments 1-11, whereinthe one or more amino acid modifications are selected from amongN52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P, N52S/Y146C/Y152C, N52H/C198R,N52H/C140D/T225A, N52H/C198R/T225A, N52H/K92R, N52H/S99G, N57Y/Q100P,N52S/S130G/Y152C, N52S/Y152C, N52S/C198R, N52Y/N57Y/Y152C,N52Y/N57Y/H129P/C198R, N52H/L161P/C198R, N52S/T113E, N52D/S54P,N52K/L208P, N52S/Y152H, N52D/V151A, N52H/I143T, N52S/L80P,F120S/Y152H/N201S, N52S/R75Q/L203P, N52S/D158G, N52D/Q133H,N52S/N57Y/H94D/L96F/L98F/Q100R,N52S/N57Y/H94D/L96F/L98F/Q100R/G103E/F120S, N52H/F78L/Q100R,N52H/N57Y/Q100R/V110D, N52H/N57Y/R75Q/Q100R/V110D, N52H/N57Y/Q100R,N52H/N57Y/L74Q/Q100R/V110D, N52H/Q100R, N52H/S121G,A20V/N52H/N57Y/Q100R/S109G, N52H/N57Y/R61S/Q100R/V110D/L173S,N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S, N52H/N57Y, N52S/F120S,N52S/V97A, N52S/G72R, N52S/A71T/A117T, N52S/E220G,Y47H/N52S/V107A/F120S, N52H/N57Y/Q100R/V110D/S132F/M175T,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R,Q37R/N52H/N57Y/Q100R/V110N/S142F/C198R/D217V/R221G,N52H/N57Y/Q100R/V110D/C198R,N52H/N57Y/Q100R/V110D/V116A/L161M/F172S/S192G/C198R,F27S/N52H/N57Y/V110N, N52S/H94E/L961/S109N/L166Q,S18R/N52S/F93L/1143V/R221G, A20T/N52D/Y146C/Q164L,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T,N52S/H94E/L96I/V122M, N52H/N57Y/H94E/L96I/F120I/S126T/W153R/I218N,M10V/S18R/N30D/N52S/S126R/T139S/L203F, S25G/N30D/N52S/F120S/N227K,N30D/N52S/L67P/Q100K/D217G/R221K/T225S,N52H/N57Y/Q100R/V110D/A117T/T190S/C198R,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R,N52H/N57Y/V110A/C198R/R221I,M10I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M,C198R,N52H/N57Y/R61C/Y62F/Q100R/V110N/F120S/C198R,N52H/N57Y/Q100R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/N144D/F172S/C198R, N52S/H94E/L98F/Q100R,N52S/E90A, N30D/K42E/N52S, N52S/F120S/I143V/I224V,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/S54P, T38P/N52S/N57D, E11ldel, Y33del, N52H/C140del/T225A, N52H/F78L/Q100R/C198R,N52H/N57Y/R75Q/Q100P/V110D, N52H/N57Y/L74Q/V110D/S192G,N52H/S121G/C198R, N52S/F120S/N227K, N52S/A71T/A117T/T190A/C198R,T43A/N52H/N57Y/L74Q/D89G/V110D/F172S, N52H/N57Y/Q100R/V110D/S132F/M175T,N52D, N52H/N57Y/Q100R/V107I/V110D/I154F/C198R/R221G, N52Q/N207Q,N168Q/N207Q, N52Q/N168Q, N84Q/N207Q, N155Q/N207Q, N119Q/N168Q,N119Q/N207Q, N119Q/N155Q, N52Q/N84Q, N52Q/N119Q, N84Q/N119Q,N52Q/N84Q/N168Q, N52Q/N84Q/N207Q, N84Q/N155Q/N168Q, N84Q/N168Q/N207Q,N84Q/N155H/N207Q, N155Q/N168Q/N207Q, N119Q/N155Q/N168Q,N119Q/N168Q/N207Q, N84Q/N119Q/N207Q, N119Q/N155H/N207Q,N84Q/N119Q/N155Q, N52Q/N119Q/N155Q, N52H/N84Q/N119Q, N52H/N84Q,N52H/N84Q/N168Q, N52H/N84Q/N207Q, N52H/N84Q/N168Q/N207Q,N52Q/N84Q/N155Q, N52Q/N84Q/N168Q, N52Q/N84Q/N155Q/N168Q,N52Q/N84Q/N119Q/N168Q, N84Q/N119Q/N155Q/N168Q, N84Q/N155Q/N168Q/N207Q,N84Q/N119Q/N155Q/N207Q, N52Q/N84Q/N119Q/N207Q, N52Q/N84Q/N119Q/N155Q,N52Q/N84Q/N119Q/N155Q/N207Q, N84Q/N119Q/N155Q/N168Q/N207Q, F138L/L203P,N52Y/F138L/L203P, N57Y/Q100R/C198R, N57Y/F138L/L203P, Q100R/F138L,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R,N52H/N57Y/Q100R/L102R/H115R/F172S/C198R, N52H/V122A/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/N194D, N52H/N57Y/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/H115R, N52H/N57Y/Q100R/H115R,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/F172S, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/E90A/H115R, N30D/K42E N52S/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N52S/H115R/F120S/I143V/C198R, N52S/H115R/F172S/C198R,N52H/N57Y/Q100P/C198R, N52H/N57Y/Q100P H115R/F172S/C198R,N52H/N57Y/Q100P/F172S/C198R, N52H/N57Y/Q100P/H115R,N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R, N52H/Q100R/H115R/F172S,N52H/Q100R/ F172S/C198R, N52H/Q100R/H115R/F172S/C198R, orN52H/N57Y/Q100R/F172S/C198R.

13. The variant ICOSL polypeptide of any of embodiments 1-12, whereinthe one or more amino acid modifications correspond are at a position(s)selected from 52, 57, 100, 110, or 198.

14. The variant ICOSL polypeptide of any of embodiments 1-13, whereinthe one or more amino acid modifications are selected from N52H, N52D,N52Q, N52S, N52K, S54A, S54P, N57Y, Q100P, Q100R, V110A, V110D, C198R,or a conservative amino acid substitution thereof.

15. The variant ICOSL polypeptide of any of embodiments 1-14, furthercomprising one or more amino acid modifications selected from V11E,E16V, N30D, K42E, N52H, N52S, N52Y, N57Y, E90A, H94E, L96I, L98F, Q100R,Q100P, V110A, V110D, H115R, F120S, V122A, F138L, I143V, K156M, K156R,F172S, N194D, C198R, L203P, V210A, R221I, I224V, or a conservative aminoacid substitution thereof.

16. The variant ICOSL polypeptide of any of embodiments 1-14, whereinthe one or more amino acid modifications are N52H/N57Y/Q100R/C198R,N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S, N52Y/N57Y/F138L/L203P,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52H/N57Y/Q100R, N52H/Q100R,N52H/N57Y/Q100R/V110D/C198R/S212G,E16V/N52H/N57Y/Q100R/V110D/H115R/V152C/K156M/C198R, N30D/K42E/N52S,N52S/F120S/I143V/I224V, N52S/E90A, N52H/N57Y/V110A/C198R/R221I,N52H/N57Y/Q100P, or N52S/N194D.

17. The variant ICOSL polypeptide of any of embodiments 1-16, whereinthe variant ICOSL polypeptide comprises the IgV domain or a specificfragment thereof and the IgC domain or a specific fragment thereof.

18. The variant ICOSL polypeptide of any of embodiments 1-17, comprisingthe sequence of amino acids set forth in any of SEQ ID NOS: 109-142,239, 280-325, 364-381, 387-424, 427-433, 435-470 or a specific bindingfragment thereof, or a sequence of amino acids that exhibits at least95% sequence identity to any of SEQ ID NOS: 109-142, 239, 280-325,364-381, 387-424, 427-433, 435-470 or a specific binding fragmentthereof and that contains the one or more of the amino acidsubstitutions.

19. The variant ICOSL polypeptide of any of embodiments 1-18, whereinthe variant ICOSL polypeptide comprises the IgV domain or a specificbinding fragment thereof.

20. The variant ICOSL polypeptide of any of embodiments 1-19, whereinthe IgV domain or specific binding fragment thereof is the only ICOSLportion of the variant ICOSL polypeptide.

21. The variant ICOSL polypeptide of any of embodiments 1-18, whereinthe IgC domain or specific binding fragment thereof is the only ICOSLportion of the variant ICOSL polypeptide.

22. The variant ICOSL polypeptide of any of embodiments 1-20, comprisingthe sequence of amino acids set forth in any of SEQ ID NOS: 197-199,201-208, 210, 212, 240, 326-340, 382-386, 425-426, and 434 or a specificbinding fragment thereof, a sequence of amino acids that exhibits atleast 95% sequence identity to any of SEQ ID NOS: 197-199, 201-208, 210,212, 240,326-340, 382-386, 425-426, and 434 or a specific bindingfragment thereof and that contains the one or more of the amino acidsubstitutions.

23. The variant ICOSL polypeptide of any of embodiments 1-22, whereinthe variant ICOSL polypeptide specifically binds to the ectodomain ofICOS, CD28 or CTLA-4 with increased affinity compared to the unmodifiedICOSL.

24. The variant ICOSL polypeptide of any of embodiments 1-23, whereinthe variant ICOSL polypeptide specifically binds to the ectodomain ofICOS or CD28 with increased affinity compared to the unmodified ICOSL.

25. The variant ICOSL polypeptide of any of embodiments 1-24, whereinthe variant ICOSL polypeptide specifically binds to the ectodomain ofICOS and the ectodomain of CD28 each with increased affinity compared tothe unmodified ICOSL.

26. The variant ICOSL polypeptide of any of embodiments 1-24, whereinthe variant ICOSL polypeptide specifically binds to the ectodomain ofCD28 with increased affinity compared to the unmodified ICOSL.

27. The variant ICOSL polypeptide of any of embodiments 1-26, whereinthe increased affinity to the ectodomain of CD28 is increased more than1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold , 50-fold or 60-foldcompared to the unmodified ICOSL.

28. The variant ICOSL polypeptide of any of embodiments 1-24, whereinthe variant ICOSL polypeptide specifically binds to the ectodomain ofICOS with increased affinity compared to the unmodified ICOSL.

29. The variant ICOSL polypeptide of any of embodiments 1-25 and 28,wherein the increased affinity to the ectodomain of ICOS is increasedmore than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 40-fold, 50-fold, 60-fold or70-fold compared to the unmodified ICOSL.

30. The variant ICOSL polypeptide of any of embodiments 1-29, whereinthe one or more amino acid modifications correspond to position(s)selected from 52, 54 or 57.

31. The variant ICOSL polypeptide of any of embodiments 1-30, whereinthe one or more amino acid modifications are selected from N52H, N52D,N52S, N52Y, N52K, S54A, S54P, N57Y or a conservative amino acidsubstitution thereof.

32. The variant ICOSL polypeptide of any of embodiments 1-31, whereinthe one or more amino acid modifications correspond to position(s)selected from 52 or 57.

33. The variant ICOSL polypeptide of any of embodiments 1-31, whereinthe one or more amino acid modifications are selected from N52H, N52D,N52S, N52K or N57Y.

34. The variant ICOSL polypeptide of any of embodiments 25-29 andembodiment 33, further comprising one or more amino acid modificationsselected from N52H, N52D, N52S, N52Y, N52K, S54A, S54P, N57Y, R75Q,L80P, K92R, S99G, H94D, L96F, L98F, S99G, Q100R, Q100P, G103E, T113E,F120S, H129P, S130G, Q133H, F138L, C140D, C140del, I143T, Y146C, V151A,Y152C, Y152H, D158G, L161P, C198R, N201S, L203P, L208P or T225A, or aconservative amino acid substitution thereof.

35. The variant ICOSL polypeptide of any of embodiments 1-34, whereinthe one or more amino acid modifications are selected from amongN52Y/N57Y/F138L/L203P, N52H/N57Y/Q100P, N52S/Y146C/Y152C, N52H/C198R,N52H/C140del/T225A, N52H/C198R/T225A, N52H/K92R, N57Y/Q100P, N52S/C198R,N52Y/N57Y/Y152C, N52Y/N57Y/H129P/C198R, N52H/L161P/C198R, N52S/T113E,N52S/S54P, N52K/L208P, N52S/Y152H, N52H/I143T, N52S/R75Q/L203P,N52S/D158G, N52D/Q133H, N52H/N57Y/Q100R/V110D/C198R/S212G,N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/S54P, T38P/N52S/N57D,N52H/C140del/T225A, N52H/F78L/Q100R/C198R, N52H/N57Y/R75Q/Q100P/V110D,N52H/N57Y/L74Q/V110D/S192G, N52H/S121G/C198R, N52S/F120S/N227K,N52S/A71T/A117T/T190A/C198R, T43A/N52H/N57Y/L74Q/D89G/V110D/F172S,N52H/N57Y/Q100R/V110D/S132F/M175T,N52H/N57Y/Q100R/V1071/V110D/1154F/C198R/R221G, N52Q/N207Q, N52Q/N168Q,N52Q/N84Q, N52Q/N119Q, N52Q/N84Q/N168Q, N52Q/N84Q/N207Q,N52Q/N119Q/N155Q, N52H/N84Q/N119Q, N52H/N84Q, N52H/N84Q/N168Q,N52H/N84Q/N207Q, N52H/N84Q/N168Q/N207Q, N52Q/N84Q/N155Q,N52Q/N84Q/N168Q, N52Q/N84Q/N155Q/N168Q, N52Q/N84Q/N119Q/N168Q,N52Q/N84Q/N119Q/N207Q, N52Q/N84Q/N119Q/N155Q,N52Q/N84Q/N119Q/N155Q/N207Q, N52Y/F138L/L203P, N57Y/Q100R/C198R,N57Y/F138L/L203P, N52H/N57Y/Q100R/H115R/C198R,N52H/N57Y/Q100R/F172S/C198R, N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/1143V/F172S/C198R,N52H/N57Y/Q100R/L102R/H115R/F172S/C198R, N52H/V122A/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/N194D, N52H/N57Y/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/H115R, N52H/N57Y/Q100R/H115R,N52H/N57Y/Q100R/H115R/F172S/1224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/F172S, N52H/Q100R/H115R/1143T/F172S,N52H/N57Y/Q100P/H115R/F172S, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/E90A/H115R, N30D/K42E/N52S/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N52S/H115R/F120S/1143V/C198R, N52S/H115R/F172S/C198R,N52H/N57Y/Q100P/C198R, N52H/N57Y/Q100P H115R/F172S/C198R,N52H/N57Y/Q100P/F172S/C198R, N52H/N57Y/Q100P/H115R,N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R, N52H/Q100R/H115R/F172S,N52H/Q100R/ F172S/C198R, N52H/Q100R/H115R/F172S/C198R, orN52H/N57Y/Q100R/F172S/C198R.

36. The variant ICOSL polypeptide of any of embodiments 1-35, whereinthe one or more amino acid modifications are selected fromN52H/N57Y/F138L/L203P, N52H/N57Y/Q100P, N52H/K92R, N52H/C140del/T225A,N52H/C198R/T225A, N52H/K92R, N57Y/Q100P, N52Y/N57Y/H129P/C198R,N52H/L161P/C198R, N52K/L208P or N52H/I143T.

37. The variant ICOSL polypeptide of any of embodiments 1-34, whereinthe one or more amino acid substitutions are selected from N57Y/Q100P,N52S/S130G/Y152C, N52S/Y152C, N52Y/N57Y/Y152C, N52H/L161P/C198R,N52H/L161P/C198R, N52S/L80P, A20V/N52H/N57Y/Q100R/S109G,N52H/N57Y/R61S/Q100R/V110D/L173S,N52H/N57Y/Q100R/V107I/V110D/S132F/I154F/C198R/R221G,Q37R/N52H/N57Y/Q100R/V110N/S142F/C198R/D217V/R221G,N52H/N57Y/Q100R/V110D/C198R, F27S/N52H/N57Y/V110N,S18R/N52S/F93L/I143V/R221G, A20T/N52D/Y146C/Q164L,N52H/N57Y/H94E/L96I/F120I/S126T/W153R/I218N,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R, orM10I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M/C198R.

38. The variant ICOSL polypeptide of any of embodiments 1-37, whereinthe variant polypeptide specifically binds to the ectodomain of ICOS,CD28 or CTLA4 with increased selectivity compared to the unmodifiedICOSL.

39. The variant ICOSL polypeptide of embodiment 38, wherein theincreased selectivity comprises a greater ratio of binding of thevariant polypeptide for one cognate binding partner selected from amongICOS, CD28 and CTLA4 versus another of the cognate binding partnercompared to the ratio of binding of the unmodified ICOSL polypeptide forthe one cognate binding partner versus the another of the cognatebinding partner.

40. The variant ICOSL polypeptide of embodiment 39, wherein the ratio isgreater by at least or at least about 1.5-fold, 2.0-fold, 3.0-fold,4.0-fold. 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-foldor more.

41. The variant ICOSL polypeptide of any of embodiments 1-40, whereinthe variant ICOSL polypeptide specifically binds to the ectodomain ofICOS or CD28 with increased affinity and specifically binds to theectodomain of the other of ICOS or CD28 with decreased affinity comparedto the unmodified ICOSL.

42. The variant ICOSL polypeptide of embodiment 41, wherein the variantICOSL polypeptide specifically binds to the ectodomain of ICOS withincreased affinity and specifically binds to the ectodomain of CD28 withdecreased affinity compared to the unmodified ICOSL.

43. The variant ICOSL polypeptide of embodiment 41, wherein the variantICOSL polypeptide specifically binds to the ectodomain of CD28 withincreased affinity and specifically binds to the ectodomain of ICOS withdecreased affinity compared to the unmodified ICOSL.

44. The variant ICOSL polypeptide of any of embodiments 1-43, comprisingthe amino acid substitutions N52S/R75Q/L203P or N30D/K42E/N52S.

45. The variant ICOSL polypeptide of any of embodiments 1-40, whereinthe variant ICOSL polypeptide specifically binds to the ectodomain ofCTLA-4 with increased affinity compared to the unmodified ICOSL.

46. The variant ICOSL polypeptide of embodiment 45, wherein theincreased affinity to the ectodomain of CTLA-4 is increased more than1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 20-fold, 40-fold, 50-fold, 60-fold or 70-foldcompared to the unmodified ICOSL.

47. The variant ICOSL polypeptide of any of embodiments 8-46, whereinthe ICOS is a human ICOS.

48. The variant ICOSL polypeptide of any of embodiments 8-47, whereinthe CD28 is a human CD28.

49. The variant ICOSL polypeptide of any of embodiments 1-48, whereinthe binding is altered (increased or decreased) more than 1.2-fold,1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 30-fold 40-fold or 50-fold compared to theunmodified ICOSL.

50. The variant ICOSL polypeptide of any of embodiments 1-49 that is asoluble protein. 51. The variant ICOSL polypeptide of any of embodiments1-50 that is linked to a multimerization domain.

52. The variant ICOSL polypeptide of any of embodiments 1-51, whereinthe variant ICOSL polypeptide is a multimeric polypeptide, optionally adimeric polypeptide, comprising a first variant ICOSL polypeptide linkedto a multimerization domain and a second variant ICOSL polypeptidelinked to a multimerization domain.

53. The variant ICOSL polypeptide of embodiment 52, wherein the firstvariant ICOSL polypeptide and the second variant ICOSL polypeptide arethe same or different.

54. The variant ICOSL polypeptide of any of embodiments 51-53, whereinthe multimerization domain is an Fc domain or a variant thereof withreduced effector function.

55. The variant ICOSL polypeptide of any of embodiments 1-54 that islinked to a moiety that increases biological half-life of thepolypeptide.

56. The variant ICOSL polypeptide of any of embodiments 1-55 that islinked to an Fc domain or a variant thereof with reduced effectorfunction.

57. The variant ICOSL polypeptide of any of embodiments 54-56, wherein:

the Fc domain is mammalian, optionally human; or

the variant Fc domain comprises one or more amino acid modificationscompared to an unmodified Fc domain that is mammalian, optionally human.

58. The variant ICOSL polypeptide of any one of embodiments 54, 56, and57, wherein the Fc domain or variant thereof comprises the sequence ofamino acids set forth in SEQ ID NO:226 or SEQ ID NO:227 or a sequence ofamino acids that exhibits at least 85% sequence identity to SEQ IDNO:226 or SEQ ID NO:227.

59. The variant ICOSL polypeptide of any of embodiments 54 and 56-58,wherein the Fc domain comprises one or more amino acid modificationsselected from among E233P, L234A, L234V, L235A, L235E, G236del, G237A,S267K, R292C, N297G and V302C,each by EU numbering.

60. The variant ICOSL polypeptide of any of embodiments 54 and 56-59,wherein the Fc domain comprises the amino acid modification C220S by EUnumbering.

61. The variant ICOSL polypeptide of any of embodiments 54 and 56-60,wherein the Fc domain comprises the sequence of amino acids set forth inany of SEQ ID NOS:474, 476, 477, 478 or a sequence of amino acids thatexhibits at least 85% sequence identity to any of SEQ ID NOS: 474, 476,477, 478 and exhibits reduced effector function.

62. The variant ICOSL polypeptide of any of embodiments 51-61, whereinthe variant ICOSL polypeptide is linked to the multimerization domain orFc indirectly via a linker, optionally a G4S linker.

63. The variant ICOSL polypeptide of any of embodiments 1-49, that is atransmembrane immunomodulatory protein further comprising atransmembrane domain linked to the extracellular domain (ECD) orspecific binding fragment thereof of the variant ICOSL polypeptide.

64. The variant ICOSL polypeptide of embodiment 63, wherein thetransmembrane domain comprises the sequence of amino acids set forth asresidues 257-277 of SEQ ID NO:5 or a functional variant thereof thatexhibits at least 85% sequence identity to residues 257-277 of SEQ IDNO:5.

65. The variant ICOSL polypeptide of embodiment 63 or embodiment 64,further comprising a cytoplasmic signaling domain linked to thetransmembrane domain.

66. The variant ICOSL polypeptide of embodiment 65, wherein thecytoplasmic signaling domain comprises the sequence of amino acids setforth as residues 278-302 of SEQ ID NO:5 or a functional variant thereofthat exhibits at least 85% sequence identity to residues 278-302 of SEQID NO:5.

67. The variant ICOSL polypeptide of any of embodiments 63-66,comprising the sequence of amino acids set forth in any of SEQ IDNOS:494-503 or a sequence of amino acids that exhibits at least 85%sequence identity to any of SEQ ID NOS:494-503.

68. The variant ICOSL polypeptide of any of embodiments 1-67, whereinthe variant ICOSL increases IFN-gamma (interferon-gamma) expressionrelative to the unmodified ICOSL in an in vitro primary T-cell assay.

69. The variant ICOSL polypeptide of any of embodiments 1-68, whereinthe variant ICOSL decreases IFN-gamma (interferon-gamma) expressionrelative to the unmodified ICOSL in an in vitro primary T-cell assay.

70. The variant ICOSL polypeptide of any of embodiments 1-51 that isdeglycosylated.

71. An immunomodulatory protein, comprising the variant ICOSLpolypeptide of any of embodiments 1-70 linked to a second polypeptidecomprising an immunoglobulin superfamily (IgSF) domain.

72. The immunomodulatory protein of embodiment 71, wherein the IgSFdomain is affinity modified and exhibits altered binding to one or moreof its cognate binding partner(s) compared to the unmodified orwild-type IgSF domain.

73. The immunomodulatory polypeptide of embodiment 72, wherein the IgSFdomain exhibits increased binding to one or more of its cognate bindingpartner(s) compared to the unmodified or wild-type IgSF domain.

74. The immunomodulatory polypeptide of any one of embodiments 71-73,wherein the variant ICOSL polypeptide is a first ICOSL variantpolypeptide and the IgSF domain of the second polypeptide is an IgSFdomain from a second variant ICOSL polypeptide of any of embodiments1-70, wherein the first and second ICOSL variant are the same ordifferent.

75. The immunomodulatory protein of any one of embodiments 71-74,wherein the variant ICOSL polypeptide is capable of specifically bindingto CD28 or ICOS and the IgSF domain of the second polypeptide is capableof binding to a cognate binding partner other than one specificallybound by the ICOSL variant polypeptide.

76. The immunomodulatory polypeptide of embodiment 75, wherein the IgSFdomain is from a member of the B7 family.

77. The immunomodulatory polypeptide of any of embodiments 71-73 and 75,wherein the IgSF domain is a tumor-localizing moiety that binds to aligand expressed on a tumor.

78. The immunomodulatory polypeptide of embodiment 77, wherein theligand is B7H6.

79. The immunomodulatory polypeptide of embodiment 77 or embodiment 78,wherein the IgSF domain is from NKp30.

80. The immunomodulatory polypeptide of any of embodiments 71-79,wherein the IgSF domain is or comprises an IgV domain.

81. The immunomodulatory polypeptide of any of embodiments 71-80,wherein the variant ICOSL polypeptide is or comprise an IgV domain.

82. The immunomodulatory protein of any of embodiments 71-81, whereinthe immunomodulatory protein comprises a multimerization domain linkedto one or both of the variant ICOSL polypeptide or the secondpolypeptide comprising the IgSF domain.

83. The immunomodulatory protein of embodiment 82, wherein themultimerization domain is an Fc domain or a variant thereof with reducedeffector function.

84. The immunomodulatory protein of any of embodiments 71-83 that isdimeric.

85. The immunomodulatory protein of embodiment 84 that is homodimeric.

86. The immunomodulatory protein of embodiment 84 that is heterodimeric.

87. A conjugate, comprising a variant ICOSL polypeptide of any ofembodiments 1-70 or an immunomodulatory polypeptide of any ofembodiments 71-86 linked to a moiety.

88. The conjugate of embodiment 87, wherein the moiety is a targetingmoiety that specifically binds to a molecule on the surface of a cell.

89. The conjugate of embodiment 88, wherein the targeting moietyspecifically binds to a molecule on the surface of an immune cell.

90. The conjugate of embodiment 89, wherein the immune cell is anantigen presenting cell or a lymphocyte.

91. The conjugate of embodiment 88, wherein the targeting moiety is atumor-localizing moiety that binds to a molecule on the surface of atumor.

92. The conjugate of any of embodiments87-91, wherein the moiety is aprotein, a peptide, nucleic acid, small molecule or nanoparticle.

93. The conjugate of any of embodiments 87-92, wherein the moiety is anantibody or antigen-binding fragment.

94. The conjugate of any of embodiments 87-93, wherein the conjugate isdivalent, tetravalent, hexavalent or octavalent.

95. A nucleic acid molecule(s), encoding a variant ICOSL polypeptide ofany of embodiments 1-70 or an immunomodulatory polypeptide of any ofembodiments 71-86.

96. The nucleic acid molecule(s) of embodiment 95 that is syntheticnucleic acid.

97. The nucleic acid molecule(s) of embodiment 95 or embodiment 96 thatis cDNA.

98. A vector, comprising the nucleic acid molecule(s) of any ofembodiments 95-97.

99. The vector of embodiment 98 that is an expression vector.

100. The vector of embodiment 98 or embodiment 99, wherein the vector isa mammalian expression vector or a viral vector.

101. A cell, comprising the vector of embodiment 102 or embodiment 103.

102. The cell of embodiment 101 that is a mammalian cell.

103. The cell of embodiment 101 or embodiment 102 that is a human cell.

104. A method of producing a variant ICOSL polypeptide or animmunomodulatory protein, comprising introducing the nucleic acidmolecule of any of embodiments 95-97 or vector of any of embodiments98-100 into a host cell under conditions to express the protein in thecell.

105. The method of embodiment 104, further comprising isolating orpurifying the variant ICOSL polypeptide or immunomodulatory protein fromthe cell.

106. A method of engineering a cell expressing a variant ICOSL variantpolypeptide, comprising introducing a nucleic acid molecule encoding thevariant ICOSL polypeptide of any of embodiments 1-70 or immunomodulatorypolypeptide of any of embodiments 71-86 into a host cell underconditions in which the polypeptide is expressed in the cell.

107. An engineered cell, comprising the variant ICOSL polypeptide of anyof embodiments 1-70, the immunomodulatory polypeptide of any ofembodiments 71-86, the nucleic acid molecule of any of embodiments 95-97or the vector of any of embodiments 98-100.

108. The engineered cell of embodiment 107, wherein the variant ICOSLpolypeptide or immunomodulatory polypeptide comprises a signal peptide.

109. The engineered cell of embodiment 107 or embodiment 108, whereinthe variant ICOSL polypeptide or immunomodulatory polypeptide does notcomprise a transmembrane domain and/or is not expressed on the surfaceof the cell.

110. The engineered cell of any of embodiments 107-109, wherein thevariant ICOSL polypeptide or immunomodulatory polypeptide is secretedfrom the engineered cell.

111. The engineered cell of embodiment 107 or embodiment 108, whereinthe engineered cell comprises a variant ICOSL polypeptide that comprisesa transmembrane domain and/or is the transmembrane immunomodulatoryprotein of any of embodiments 63-67.

112. The engineered cell of embodiment 107, embodiment 108 or embodiment111, wherein the variant ICOSL polypeptide is expressed on the surfaceof the cell.

113. The engineered cell of embodiment 112, wherein the cell is animmune cell.

114. The engineered cell of embodiment 113, wherein the immune cell isan antigen presenting cell (APC) or a lymphocyte.

115. The engineered cell of any of embodiments 107-114 that is a primarycell.

116. The engineered cell of any of embodiments 107-115, wherein the cellis a mammalian cell.

117. The engineered cell of any of embodiments 107-116, wherein the cellis a human cell.

118. The engineered cell of any of embodiments 107-117, wherein thelymphocyte is a T cell.

119. The engineered cell of embodiment 114, wherein the APC is anartificial APC.

120. The engineered cell of any of embodiments 107-119, furthercomprising a chimeric antigen receptor (CAR) or an engineered T-cellreceptor.

121. An infectious agent, comprising a nucleic acid molecule encoding avariant ICOSL polypeptide of any of embodiments 1-70 or animmunomodulatory polypeptide of any of embodiments 71-86.

122. The infectious agent of embodiment 121, wherein the encoded variantICOSL polypeptide or immunomodulatory polypeptide does not comprise atransmembrane domain and/or is not expressed on the surface of a cell inwhich it is expressed.

123. The infectious agent of embodiment 121 or embodiment 122, whereinthe encoded variant ICOSL polypeptide or immunomodulatory polypeptide issecreted from a cell in which it is expressed.

124. The infectious agent of embodiment 121, wherein the encoded variantICOSL polypeptide comprises a transmembrane domain.

125. The engineered cell of embodiment 107, embodiment 108 or embodiment111, wherein the encoded variant ICOSL polypeptide is expressed on thesurface of a cell in which it is expressed.

126. The infectious agent of any of embodiments 121-125, wherein theinfectious agent is a bacteria or a virus.

127. The infectious agent of embodiment 126, wherein the virus is anoncolytic virus.

128. The infectious agent of embodiment 127, wherein the oncolytic virusis an adenoviruses, adeno-associated viruses, herpes viruses, HerpesSimplex Virus, Vesticular Stomatic virus, Reovirus, Newcastle Diseasevirus, parvovirus, measles virus, vesticular stomatitis virus (VSV),Coxsackie virus or a Vaccinia virus.

129. The infectious agent of embodiment 126, wherein the virusspecifically targets dendritic cells (DCs) and/or is dendriticcell-tropic.

130. The infectious agent of embodiment 129, wherein the virus is alentiviral vector that is pseudotyped with a modified Sindbis virusenvelope product.

131. The infectious agent of any of embodiments 121-130, furthercomprising a nucleic acid molecule encoding a further gene product thatresults in death of a target cell or that can augment or boost an immuneresponse.

132. The infectious agent of embodiment 131, wherein the further geneproduct is selected from an anticancer agent, anti-metastatic agent, anantiangiogenic agent, an immunomodulatory molecule, an immune checkpointinhibitor, an antibody, a cytokine, a growth factor, an antigen, acytotoxic gene product, a pro-apoptotic gene product, an anti-apoptoticgene product, a cell matrix degradative gene, genes for tissueregeneration or a reprogramming human somatic cells to pluripotency.

133. A pharmaceutical composition, comprising the variant ICOSLpolypeptide of any of embodiments 1-70, an immunomodulatory protein ofany of embodiments 71-86, a conjugate of any of embodiments 87-94, anengineered cell of any of embodiments 107-120 or an infectious agent ofany of embodiments 121-132.

134. The pharmaceutical composition of embodiment 133, comprising apharmaceutically acceptable excipient.

135. The pharmaceutical composition of embodiment 123 or 134, whereinthe pharmaceutical composition is sterile.

136. An article of manufacture comprising the pharmaceutical compositionof any of embodiments 133-135 in a vial.

137. The article of manufacture of embodiment 136, wherein the vial issealed.

138. A kit comprising the pharmaceutical composition of any ofembodiments 133-135, and instructions for use.

139. A kit comprising the article of manufacture according to embodiment136 and 137, and instructions for use.

140. A method of modulating an immune response in a subject, comprisingadministering the pharmaceutical composition of any of embodiments133-135 to the subject.

141. A method of modulating an immune response in a subject, comprisingadministering the engineered cells of any of embodiments 107-120.

142. The method of embodiment 141, wherein the engineered cells areautologous to the subject.

143. The method of embodiment141, wherein the engineered cells areallogenic to the subject.

144. The method of any of embodiments 140-143, wherein modulating theimmune response treats a disease or condition in the subject.

145. The method of any of embodiments 140-144, wherein the immuneresponse is increased.

146. The method of any of embodiments 140, 144 and 145, wherein animmunomodulatory protein or conjugate comprising a variant ICOSLpolypeptide linked to a tumor-localizing moiety is administered to thesubject.

147. The method of embodiment 146, wherein the tumor-localizing moietyis or comprises a binding molecule that recognizes a tumor antigen.

148. The method of embodiment 147, wherein the binding moleculecomprises an antibody or an antigen-binding fragment thereof orcomprises a wild-type IgSF domain or variant thereof.

149. The method of any of embodiments 140and 144-148, wherein apharmaceutical composition comprising the immunomodulatory protein ofany of embodiments 77-86 or the conjugate of any of embodiments 87-94 isadministered to the subject.

150. The method of any of embodiments 140-145, wherein an engineeredcell comprising a variant ICOSL polypeptide that is a transmembraneimmunomodulatory protein is administered to the subject and/or theengineered cell of 107, 108 and 111-120.

151. The method of any of embodiments 140, 144 and 145, wherein aninfectious agent encoding a variant ICOSL polypeptide that is atransmembrane immunomodulatory protein is administered to the subject,optionally under conditions in which the infectious agent infects atumor cell or immune cell and the transmembrane immunomodulatory proteinis expressed on the surface of the infected cell.

152. The method of any of embodiment 150 or embodiment 151, wherein thetransmembrane immunomodulatory protein is of any of embodiments 63-70.

153. The method of any of embodiments 140-152, wherein the disease orcondition is a tumor or cancer.

154. The method of any one of embodiments 140-153, wherein the diseaseor condition is selected from melanoma, lung cancer, bladder cancer, ahematological malignancy, liver cancer, brain cancer, renal cancer,breast cancer, pancreatic cancer, colorectal cancer, spleen cancer,prostate cancer, testicular cancer, ovarian cancer, uterine cancer,gastric carcinoma, a musculoskeletal cancer, a head and neck cancer, agastrointestinal cancer, a germ cell cancer, or an endocrine andneuroendocrine cancer.

155. The method of any of embodiments 140-144, wherein the immuneresponse is decreased.

156. The method of any of embodiments 140, 144 and 155, wherein avariant ICOSL polypeptide or immunomodulatory protein that is soluble isadministered to the subject.

157. The method of embodiment 156, wherein the soluble polypeptide orimmunomodulatory protein is an Fc fusion protein.

158. The method of any of embodiments 140, 144 and 155-157, wherein apharmaceutical composition comprising a variant ICOSL polypeptide of anyof embodiments 1-62 and 68-70, or the immunomodulatory protein of any ofembodiments 71-76 and 80-86 is administered to the subject.

159. The method of any of embodiments 140-144 and 155-157, wherein anengineered cell comprising a secretable variant ICOSL polypeptide isadministered to the subject.

160. The method of any of embodiments 140-144, 155-157 and 159, whereinan engineered cell of any of embodiments 107-110 and 113-120 isadministered to the subject.

161. The method of any of embodiments 140, 144 and 155-157 and 159,wherein an infectious agent encoding a variant ICOSL polypeptide that isa secretable immunomodulatory protein is administered to the subject,optionally under conditions in which the infectious agent infects atumor cell or immune cell and the secretable immunomodulatory protein issecreted from the infected cell.

162. The method of any of embodiments 140-144 and 155-161, wherein thedisease or condition is an inflammatory or autoimmune disease orcondition.

163. The method of any of embodiments 140-144 and 155-162, wherein thedisease or condition is an Antineutrophil cytoplasmic antibodies(ANCA)-associated vasculitis, a vasculitis, an autoimmune skin disease,transplantation, a Rheumatic disease, an inflammatory gastrointestinaldisease, an inflammatory eye disease, an inflammatory neurologicaldisease, an inflammatory pulmonary disease, an inflammatory endocrinedisease, or an autoimmune hematological disease.

164. The method of embodiment 162 or embodiment 163, wherein the diseaseor condition is selected from inflammatory bowel disease, transplant,Crohn's disease, ulcerative colitis, multiple sclerosis, asthma,rheumatoid arthritis, or psoriasis.

165. The variant ICOSL polypeptide of any of embodiments 1-70, whereinthe amino acid modification is an amino acid substitution, insertion ordeletion.

IX. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Example 1 Generation of Mutant DNA Constructs of IgSF Domains

Example 1 describes the generation of mutant DNA constructs of humanICOSL IgSF domains for translation and expression on the surface ofyeast as yeast display libraries.

A. Degenerate Libraries

Constructs were generated based on a wildtype human ICOSL sequence ofthe extracellular domain (ECD) set forth in SEQ ID NO:32 (containing theECD domain corresponding to residues 19-256 as set forth in UniProtAccession No. 075144.2) as follows:

DTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKITENPVSTGEKNAAT

For libraries that target specific residues for complete or partialrandomization with degenerate codons, the coding DNA encoding SEQ IDNO:32 was ordered from Integrated DNA Technologies (Coralville, Iowa) asa set of overlapping oligonucleotides of up to 80 base pairs (bp) inlength. To generate a library of diverse variants of the ECD, theoligonucleotides contained desired degenerate codons at desired aminoacid positions. Degenerate codons were generated using an algorithm atthe URL: rosettadesign.med.unc.edu/SwiftLib/.

In general, positions to mutate and degenerate codons were chosen fromhomology models (ICOSL) of the target-ligand pairs of interest toidentify ligand contact residues as well as residues that are at theprotein interaction interface. This analysis was performed using astructure viewer available at the URL: spdbv.vital-it.ch).

The next step in library design was the alignment of human, mouse, ratand monkey ICOSL sequences to identify conserved residues. Based on thisanalysis, conserved target residues were mutated with degenerate codonsthat only specified conservative amino acid changes plus the wild-typeresidue. Residues that were not conserved, were mutated moreaggressively, but also including the wild-type residue. Degeneratecodons that also encoded the wild-type residue were deployed to avoidexcessive mutagenesis of target protein. For the same reason, only up to20 positions were targeted for mutagenesis at a time. These residueswere a combination of contact residues and non-contact interfaceresidues.

The oligonucleotides were dissolved in sterile water, mixed in equimolarratios, heated to 95° C. for five minutes and slowly cooled to roomtemperature for annealing. ECD-specific oligonucleotide primers thatanneal to the start and end of the ECDs, respectively, were then used togenerate PCR product. ECD-specific oligonucleotides which overlap by40-50bp with a modified version of pBYDS03 cloning vector (LifeTechnologies USA), beyond and including the BamHI and KpnI cloningsites, were then used to amplify 100 ng of PCR product from the priorstep to generate a total of 5 μg of DNA. Both PCR's were by polymerasechain reaction (PCR) using OneTaq 2×PCR master mix (New England Biolabs,USA). The second PCR products were purified using a PCR purification kit(Qiagen, Germany) and resuspended in sterile deionized water.

To prepare for library insertion, a modified yeast display version ofvector pBYDS03 was digested with BamHI and KpnI restriction enzymes (NewEngland Biolabs, USA) and the large vector fragment was gel-purified anddissolved in sterile, deionized water. Electroporation-ready DNA for thenext step was generated by mixing 12 μg of library DNA with 4 μg oflinearized vector in a total volume of 50 μl deionized and sterilewater. An alternative way to generate targeted libraries, was to carryout site-directed mutagenesis (Multisite kit, Agilent, USA) of thetarget ECD with oligonucleotides containing degenerate codons. Thisapproach was used to generate sublibraries that only target specificstretches of target protein for mutagenesis. In these cases,sublibraries were mixed before proceeding to the selection steps. Ingeneral, library sizes were in the range of 10E7 to 10E8 clones, exceptthat sublibraries were only in the range of 10E4 to 10E5. Largelibraries and sublibraries are generated for ICOSL.

B. Random Libraries

Random libraries were also constructed to identify variants of the ECDof ICOSL set forth in SEQ ID N0:32 (containing the ECD domaincorresponding to residues 19-256 as set forth in UniProt Accession No.075144.2 flanked by adjacent N- and C-terminal residues of the wildtypesequence). DNA encoding the wild-type ECD was cloned between the BamHIand KpnI sites of modified yeast display vector pBYDS03 and thenreleased using the same restriction enzymes. The released DNA was thenmutagenized with the Genemorph II kit (Agilent, USA) so as to generatean average of three to five amino acid changes per library variant.Mutagenized DNA was then amplified by the two-step PCR and furtherprocessed as described above for targeted libraries.

Example 2 Introduction of DNA Libraries into Yeast

Example 2 describes the introduction of ICOSL DNA libraries into yeast.

To introduce degenerate and random library DNA into yeast,electroporation-competent cells of yeast strain BJ5464 (ATCC.org; ATCCnumber 208288) were prepared and electroporated on a Gene Pulser II(Biorad, USA) with the electroporation-ready DNA from the step aboveessentially as described (Colby, D. W. et al. 2004 Methods Enzymology388, 348-358). The only exception is that transformed cells were grownin non-inducing minimal selective SCD-Leu medium to accommodate the LEU2selective marker carried by modified plasmid pBYDS03.

Library size was determined by plating dilutions of freshly recoveredcells on SCD-Leu agar plates and then extrapolating library size fromthe number of single colonies from plating that generated at least 50colonies per plate. The remainder of the electroporated culture wasgrown to saturation and cells from this culture were subcultured intothe same medium once more to minimize the fraction of untransformedcells. To maintain library diversity, this subculturing step was carriedout using an inoculum that contained at least 10× more cells than thecalculated library size. Cells from the second saturated culture wereresuspended in fresh medium containing sterile 25% (weight/volume)glycerol to a density of 10E10/ml and frozen and stored at −80° C.(frozen library stock).

One liter of SCD-Leu media consists of 14.7 grams of sodium citrate,4.29 grams of citric acid monohydrate, 20 grams of dextrose, 6.7 gramsof Difco brand yeast nitrogen base, and 1.6 grams yeast syntheticdrop-out media supplement without leucine. Media was filtered sterilizedbefore use using a 0.2 μM vacuum filter device.

Library size was determined by plating dilutions of freshly recoveredcells on SCD-Leu agar plates and then extrapolating library size fromthe number of single colonies from a plating that generate at least 50colonies per plate.

To segregate plasmid from cells that contain two or more differentlibrary clones, a number of cells corresponding to 10 times the librarysize, were taken from the overnight SCD-Leu culture and subcultured1/100 into fresh SCD-Leu medium and grown overnight. Cells from thisovernight culture were resuspended in sterile 25% (weight/volume)glycerol to a density of 10E10/ml and frozen and stored at −80° C.(frozen library stock).

Example 3 Yeast Selection

Example 3 describes the selection of yeast expressing affinity modifiedvariants of ICOSL.

A number of cells equal to at least 10 times the library size werethawed from individual library stocks, suspended to 0.1×10E6 cells/ml innon-inducing SCD-Leu medium, and grown overnight. The next day, a numberof cells equal to 10 times the library size were centrifuged at 2000 RPMfor two minutes and resuspended to 0.5×10E6 cells/ml in inducingSCDG-Leu media. One liter of the SCDG-Leu induction media consists of5.4 grams Na₂HPO₄, 8.56 grams of NaH₂PO₄.H₂O, 20 grams galactose, 2.0grams dextrose, 6.7 grams Difco yeast nitrogen base, and 1.6 grams ofyeast synthetic drop out media supplement without leucine dissolved inwater and sterilized through a 0.22 μm membrane filter device. Theculture was grown for two days at 20° C. to induce expression of libraryproteins on the yeast cell surface.

Cells were processed with magnetic beads to reduce non-binders andenrich for all ICOSL variants with the ability to bind their exogenousrecombinant counter-structure proteins. This was then followed by two tothree rounds of flow cytometry sorting using exogenous counter-structureprotein staining to enrich the fraction of yeast cells that displaysimproved binders. Magnetic bead enrichment and selections by flowcytometry are essentially as described in Keith D. Miller,1 Noah B.Pefaur,2 and Cheryl L. Bairdl Current Protocols in Cytometry4.7.1-4.7.30, July 2008.

With ICOSL libraries, target ligand proteins were sourced from R&DSystems (USA) as follows: human rCD28.Fc (i.e., recombinant CD28-Fcfusion protein), rCTLA4.Fc and rICOS.Fc. Magnetic streptavidin beadswere obtained from New England Biolabs, USA. For biotinylation ofcounter-structure protein, biotinylation kit cat#21955, LifeTechnologies, USA, was used. For two-color, flow cytometric sorting, aBecton Dickinson FACS Aria II sorter was used. ICOSL display levels weremonitored with an anti-hemagglutinin antibody labeled with Alexafluor488 (Life Technologies, USA). Ligand binding Fc fusion proteinsrCD28.Fc, rCTLA4.Fc, or rICOS.Fc were detected with PE conjugated humanIg specific goat Fab (Jackson ImmunoResearch, USA). Doublet yeast weregated out using forward scatter (FSC)/side scatter (SSC) parameters, andsort gates were based upon higher ligand binding detected in FL4 thatpossessed more limited tag expression binding in FL1.

Yeast outputs from the flow cytometric sorts were assayed for higherspecific binding affinity. Sort output yeast were expanded andre-induced to express the particular IgSF affinity modified domainvariants they encode. This population then can be compared to theparental, wild-type yeast strain, or any other selected outputs, such asthe bead output yeast population, by flow cytometry.

For ICOSL, the second sort outputs (F2) were compared to parental ICOSLyeast for binding of each rICOS.Fc, rCD28.Fc, and rCTLA4.Fc by doublestaining each population with anti-HA (hemagglutinin) tag expression andthe anti-human Fc secondary to detect ligand binding.

In the case of ICOSL yeast variants selected for binding to ICOS, the F2sort outputs gave Mean Fluorescence Intensity (MFI) values of 997, whenstained with 5.6 nM rICOS.Fc, whereas the parental ICOSL strain MFI wasmeasured at 397 when stained with the same concentration of rICOS.Fc.This represents a roughly three-fold improvement of the average bindingin this F2 selected pool of clones, and it is predicted that individualclones from that pool will have much better improved MFI/affinity whenindividually tested.

In the case of ICOSL yeast variants selected for binding to CD28, the F2sort outputs gave MFI values of 640 when stained with 100 nM rCD28.Fc,whereas the parental ICOSL strain MFI was measured at 29 when stainedwith the same concentration of rCD28.Fc (22-fold improvement). In thecase of ICOSL yeast variants selected for binding to CTLA4, the F2 sortoutputs gave MFI values of 949 when stained with 100 nM rCTLA4.Fc,whereas the parental ICOSL strain MFI was measured at 29 when stainedwith the same concentration of rCTLA4.Fc (32-fold improvement).

Importantly, the MFIs of all F2 outputs described above when measuredwith the anti-HA tag antibody on FL1 did not increase and sometimes wentdown compared to wild-type strains, indicating that increased bindingwas not a function of increased expression of the selected variants onthe surface of yeast, and validated gating strategies of only selectingmid to low expressors with high ligand binding.

Selected variant ICOSL ECD domains were further formatted as fusionproteins and tested for binding and functional activity as describedbelow.

Example 4 Reformatting Selection Outputs as Fc-Fusions and in VariousImmunomodulatory Protein Types

Example 4 describes reformatting of selection outputs asimmunomodulatory proteins containing an affinity modified (variant)extracellular domain (ECD) of ICOSL fused to an Fc molecule (variantECD-Fc fusion molecules).

Output cells from final flow cytometric ICOSL sorts were grown toterminal density in SCD-Leu medium. Plasmid DNA's from each output wereisolated using a yeast plasmid DNA isolation kit (Zymoresearch, USA).For Fc fusions, PCR primers with added restriction sites suitable forcloning into the Fc fusion vector of choice were used to batch-amplifyfrom the plasmid DNA preps the coding DNA's for the mutant target ECD's.After restriction digestion, the PCR products were ligated into anappropriate Fc fusion vector followed by chemical transformation intostrain XL1 Blue E. Coli (Agilent, USA) or NEB5alpha (New EnglandBiolabs) as directed by supplier. Exemplary of an Fc fusion vector ispFUSE-hIgG1-Fc2 (Invivogen, USA).

Dilutions of transformation reactions were plated on LB-agar containing100 μg/ml carbenicillin (Teknova, USA) to generate single colonies. Upto 96 colonies from each transformation were then grown in 96 wellplates to saturation overnight at 37° C. in LB-broth (Teknova cat #L8112) and a small aliquot from each well was submitted for DNAsequencing of the ECD insert in order to identify the mutation(s) in allclones. Sample preparation for DNA sequencing was carried out usingprotocols provided by the service provider (Genewiz; South Plainfield,N.J.). After removal of sample for DNA sequencing, glycerol was thenadded to the remaining cultures for a final glycerol content of 25% andplates were stored at −20° C. for future use as master plates (seebelow). Alternatively, samples for DNA sequencing were generated byreplica plating from grown liquid cultures to solid agar plates using adisposable 96 well replicator (VWR, USA). These plates were incubatedovernight to generate growth patches and the plates were submitted toGenewiz as specified by Genewiz. In some instances, resequencing wasperformed to verify mutations.

After identification of clones of interest from analysis ofGenewiz-generated DNA sequencing data, clones of interest were recoveredfrom master plates and individually grown to density in 5 ml liquidLB-broth containing 100 μg/ml carbenicillin (Teknova, USA) and 2 ml ofeach culture were then used for preparation of approximately 10 μg ofminiprep plasmid DNA of each clone using a standard kit such as thePureyield kit (Promega). Identification of clones of interest generallyinvolved the following steps. First, DNA sequence data files weredownloaded from the Genewiz website. All sequences were then manuallycurated so that they start at the beginning of the ECD coding region.The curated sequences were then batch-translated using a suitableprogram available at the URL: www.ebi.ac.uk/Tools/st/emboss_transeq/.The translated sequences were then aligned using a suitable programavailable at the URL: multalin.toulouse.inra.fr/multalin/multalin.html.

Clones of interest were then identified using the following criteria:1.) identical clone occurs at least two times in the alignment and 2.) amutation occurs at least two times in the alignment and preferably indistinct clones. Clones that meet at least one of these criteria wereclones that have been enriched by the sorting process due to improvedbinding.

To generate immunomodulatory proteins that are Fc fusion proteinscontaining an ECD of ICOSL with at least one affinity-modified domain(e.g. variant ICOSL ECD-Fc), the encoding nucleic acid molecule wasgenerated to encode a protein designed as follows: signal peptidefollowed by variant (mutant) ECD followed by a linker of three alanines(AAA) followed by a human IgG1 Fc containing the mutation N82G withreference to wild-type human IgG1 Fc set forth in SEQ ID NO: 226(corresponding to N297G by EU numbering). This exemplary Fc alsocontained stabilizing cysteine mutations R77C and V87C and replacementof the cysteine residue to a serine residue at position 5 (C5S), eachwith reference to wild-type human IgG1 Fc set forth in SEQ ID NO:226.(corresponding to R292C, V302C and C220S, respectively, by EUnumbering). In some cases, the NotI cloning site which contributes tothe AAA linker sequence was deleted to generate a direct fusion of theICOSL ECD and the beginning of the Fc. Since the construct does notinclude any antibody light chains that can form a covalent bond with acysteine, the human IgG1 Fc also contains replacement of the cysteineresidues to a serine residue at position 5 (C5S) compared to thewild-type or unmodified Fc set forth in SEQ ID NO: 226.

Example 5 Expression and Purification of Fc-Fusions

Example 5 describes the high throughput expression and purification ofFc-fusion proteins containing variant ECD ICOSL.

Recombinant variant Fc fusion proteins were produced with Expi293expression system (Invitrogen, USA). 4 μg of each plasmid DNA from theprevious step was added to 2000 Opti-MEM (Invitrogen, USA) at the sametime as 10.80 ExpiFectamine was separately added to another 2000Opti-MEM. After 5 minutes, the 2000 of plasmid DNA was mixed with the2000 of ExpiFectamine and was further incubated for an additional 20minutes before adding this mixture to cells. Ten million Expi293 cellswere dispensed into separate wells of a sterile 10 ml, conical bottom,deep 24 well growth plate (Thomson Instrument Company, USA) in a volume3.4 ml Expi293 media (Invitrogen, USA). Plates were shaken for 5 days at120 RPM in a mammalian cell culture incubator set to 95% humidity and 8%CO₂. Following a 5 day incubation, cells were pelleted and culturesupernatants were removed.

Protein was purified from supernatants using a high throughput 96 wellProtein A purification kit using the manufacturer's protocol (Catalognumber 45202, Life Technologies, USA). Resulting elution fractions werebuffer exchanged into PBS using Zeba 96 well spin desalting plate(Catalog number 89807, Life Technologies, USA) using the manufacturer'sprotocol. Purified protein was quantitated using 280 nm absorbancemeasured by Nanodrop instrument (Thermo Fisher Scientific, USA), andprotein purity was assessed by loading 5 μg of protein on NUPAGEpre-cast, polyacrylamide gels (Life Technologies, USA) under denaturingand reducing conditions and subsequent gel electrophoresis. Proteinswere visualized in gel using standard Coomassie staining.

Example 6 Assessment of Binding and Activity of Affinity-Matured IgSFDomain-Containing Molecules

A. Binding to Cell-Expressed Counter Structures

This Example describes Fc-fusion binding studies to show specificity andaffinity of ICOSL domain variant immunomodulatory proteins for cognatebinding partners.

To produce cells expressing cognate binding partners, full-lengthmammalian surface expression constructs for each of human CD28 and ICOSwere designed in pcDNA3.1 expression vector (Life Technologies) andsourced from Genscript, USA. Binding studies were carried out using theExpi293F transient transfection system (Life Technologies, USA)described above. The number of cells needed for the experiment wasdetermined, and the appropriate 30 ml scale of transfection wasperformed using the manufacturer's suggested protocol. For each CD28,ICOS or mock 30 ml transfection, 75 million Expi293F cells wereincubated with 30 μg expression construct DNA and 1.5 ml dilutedExpiFectamine 293 reagent for 48 hours, at which point cells wereharvested for staining.

For staining by flow cytometry, 200,000 cells of appropriate transienttransfection or negative control were plated in 96 well round bottomplates. Cells were spun down and resuspended in staining buffer (PBS(phosphate buffered saline), 1% BSA (bovine serum albumin), and 0.1%sodium azide) for 20 minutes to block non-specific binding. Afterwards,cells were centrifuged again and resuspended in staining buffercontaining 100 nM to 1 nM variant immunomodulatory protein, depending onthe experiment of each candidate CD80 variant Fc, ICOSL variant Fc, orstacked IgSF variant Fc fusion protein in 50 μl. Primary staining wasperformed on ice for 45 minutes, before washing cells in staining buffertwice. PE-conjugated anti-human Fc (Jackson ImmunoResearch, USA) wasdiluted 1:150 in 50 μl staining buffer and added to cells and incubatedanother 30 minutes on ice. Secondary antibody was washed out twice,cells were fixed in 4% formaldehyde/PBS, and samples were analyzed onFACScan flow cytometer (Becton Dickinson, USA) or a Hypercyt flowcytometer (Intellicyte, USA).

Mean Fluorescence Intensity (MFI) was calculated for each transfectantand negative parental line with Cell Quest Pro software (BectonDickinson, USA) or a Hypercyt flow cytometer (Intellicyte, USA).

B. Bioactivity Characterization

This Example further describes Fc-fusion variant protein bioactivitycharacterization in human primary T cell in vitro assays.

1. Mixed Lymphocyte Reaction (MLR)

Soluble rICOSL.Fc bioactivity was tested in a human Mixed LymphocyteReaction (MLR). Human primary dendritic cells (DC) were generated byculturing monocytes isolated from PBMC (BenTech Bio, USA) in vitro for 7days with 500U/ml rIL-4 (R&D Systems, USA) and 250U/ml rGM-CSF (R&DSystems, USA) in Ex-Vivo 15 media (Lonza, Switzerland). 10,000 maturedDC and 100,000 purified allogeneic CD4+ T cells (BenTech Bio, USA) wereco-cultured with ICOSL variant Fc fusion proteins and controls in 96well round bottom plates in 200 μl final volume of Ex-Vivo 15 media. Onday 5, IFN-gamma secretion in culture supernatants was analyzed usingthe Human IFN-gamma Duoset ELISA kit (R&D Systems, USA). Optical densitywas measured by VMax ELISA Microplate Reader (Molecular Devices, USA)and quantitated against titrated rIFN-gamma standard included in theIFN-gamma Duo-set kit (R&D Systems, USA). A second MLR protocolconsisted of human primary dendritic cells (DC) generated by culturingmonocytes isolated from PBMC (BenTech Bio, USA) in vitro for 7 days with50 ng/mL rIL-4 (R&D Systems, USA) and 80 ng/mL rGM-CSF (R&D Systems,USA) in Ex-Vivo 15 media (Lonza, Switzerland). On days 3 and 5, half ofthe media was removed and replaced with fresh media containing 50 ng/mLrIL-4 and 80 ng/mL rGM-CSF. To fully induce DC maturation,lipopolysaccharide (LPS) (InvivoGen Corp., USA) was added at 100 ng/mLto the DC cultures on day 6 and cells were incubated for an additional24 hours. Approximately, 10,000 matured DC and 100,000 purifiedallogeneic CD3+ T cells (BenTech Bio, USA) were co-cultured with ICOSLvariant Fc fusion proteins and controls in 96 well round bottom platesin 200 μl final volume of Ex-Vivo 15 media. On day 4 -5, IFN-gammasecretion in culture supernatants was analyzed using the Human IFN-gammaDuoset ELISA kit (R&D Systems, USA). Optical density was measured on aBioTek Cytation Multimode Microplate Reader (BioTek Corp., USA) andquantitated against titrated rIFN-gamma standard included in theIFN-gamma Duo-set kit (R&D Systems, USA).

2. Anti-CD3 Coimmobilization Assay

Costimulatory bioactivity of ICOSL fusion variants was determined inanti-CD3 coimmobilization assays. 1 nM or 10 nM mouse anti-human CD3(OKT3, Biolegends, USA) was diluted in PBS with 1 nM to 80nM rICOSL.Fcvariant proteins. This mixture was added to tissue culture treated flatbottom 96 well plates (Corning, USA) overnight to facilitate adherenceof the stimulatory proteins to the wells of the plate. The next day,unbound protein was washed off the plates and 100,000 purified human panT cells (BenTech Bio, US) or human T cell clone BC3 (Astarte Biologics,USA) were added to each well in a final volume of 2000 of Ex-Vivo 15media (Lonza, Switzerland). In some instances, human pan T cells werelabeled with 0.25 uM carboxyfluorescein succinimidyl ester (CFSE,ThermoFisher Scientific, USA). Cells were cultured 3 days beforeharvesting culture supernatants and measuring human IFN-gamma levelswith Duoset ELISA kit (R&D Systems, USA) as mentioned above. Cellularproliferation was determined by the percent of input cells that entereddivision as measured by CFSE dilution on cells stained withfluorescently-conjugated anti-CD4, anti-CD8 antibodies (BD, USA) ortotal T cells via flow cytometric analysis on an LSR II (BD, USA),

C. Results

Results for the binding and activity studies for exemplary testedvariants are shown in Table 7 which indicates exemplary IgSF domainamino acid substitutions (replacements) in the ECD of ICOSL selected inthe screen for affinity-maturation against the respective cognatestructures ICOS and CD28. In the Tables, the exemplary amino acidsubstitutions are designated by amino acid position number correspondingto the respective reference unmodified ECD sequence as follows. Forexample, the reference unmodified ECD sequence in Table 7 (WT ICOSL) isthe unmodified ICOSL ECD sequence set forth in SEQ ID NO:32. The aminoacid position is indicated in the middle, with the correspondingunmodified (e.g. wild-type) amino acid listed before the number and theidentified variant amino acid substitution listed after the number.Column 2 sets forth the SEQ ID NO identifier for the variant ECD foreach variant ECD-Fc fusion molecule.

Also shown is the binding activity as measured by the Mean FluorescenceIntensity (MFI) value for binding of each variant Fc-fusion molecule tocells transfected to express the cognate ligand and the ratio of the MFIcompared to the binding of the corresponding unmodified ECD-Fc fusionmolecule not containing the amino acid substitution(s) to the samecell-expressed counter structure ligand. The functional activity of thevariant Fc-fusion molecules to modulate the activity of T cells also isshown based on the calculated levels of IFN-gamma in culturesupernatants (pg/ml) generated either i) with the indicated variantECD-Fc fusion molecule coimmoblized with anti-CD3 or ii) with theindicated variant ECD-Fc fusion molecule in an MLR assay. The Table alsodepicts the ratio of IFN-gamma produced by each variant ECD-Fc comparedto the corresponding unmodified (wild-type) ECD-Fc in both functionalassays.

As shown, the selections resulted in the identification of a number ofICOSL IgSF domain variants that were affinity-modified to exhibitincreased binding for at least one, and in some cases more than one,cognate counter structure ligand. In addition, the results showed thataffinity modification of the variant molecules also exhibited improvedactivities to both increase and/or decrease immunological activitydepending on the format of the molecule. For example, coimmobilizationof the ligand likely provides a multivalent interaction with the cell tocluster or increase the avidity to favor agonist activity and increase Tcell activation compared to the unmodified (e.g. wildtype) ECD-Fcmolecule not containing the amino acid replacement(s). However, when themolecule is provided as a bivalent Fc molecule in solution, the sameIgSF domain variants exhibited an antagonist activity to decrease T cellactivation compared to the unmodified (e.g. wildtype) ECD-Fv moleculenot containing the amino acid replacement(s).

TABLE 7 ICOSL variants selected against CD28 or ICOS. Moleculesequences, binding data, and costimulatory bioactivity data. MLRCoimmobilization IFN- Binding with anti-CD3 gamma SEQ CD28 MFI IFN-gammalevels pg/ml ID NO ICOS OD (parental pg/ml (parental ICOSL mutation(s)(ECD) (parental ratio) ratio) (parental ratio) ratio) N52S 109 1.33 1621334  300 (1.55) (9.00)    (1.93)    (0.44) N52H 110 1.30 368 1268   39(1.51) (20.44)    (1.83)    (0.06) N52D 111 1.59 130 1943  190 (1.85)(7.22)    (2.80)    (0.28) N52Y/N57Y/ 112 1.02 398 510*  18 F138L/L203P(1.19) (22.11)    (1.47*)    (0.03) N52H/N57Y/Q100P 113 1.57 447 2199  25 (1.83) (24.83)    (3.18)    (0.04) N52S/Y146C/Y152C 114 1.26 391647  152 (1.47) (2.17)    (2.38)    (0.22) N52H/C198R 115 1.16 363 744*ND (1.35) (20.17)    (2.15*) (ND) N52H/C140del/ 372 ND 154 522* ND T225A(ND) (8.56)    (1.51*) (ND) N52H/C198R/T225A 117 1.41 344 778*  0 (1.64)(19.11)    (2.25*)  (0) N52H/K92R 118 1.48 347 288*  89 (1.72) (19.28)   (0.83*)    (0.13) N52H/S99G 119 0.09 29 184* 421 (0.10) (1.61)   (0.53*)    (0.61) N52Y 120 0.08 18 184* 568 (0.09) (1.00)    (0.53*)   (0.83) N57Y 121 1.40 101 580* 176 (1.63) (5.61)    (1.68*)    (0.26)N57Y/Q100P 122 0.62 285 301* 177 (0.72) (15.83)    (0.87*)    (0.26)N52S/S130G/Y152C 123 0.16 24 266* 1617 (0.19) (1.33)    (0.77*)   (2.35) N52S/Y152C 124 0.18 29 238* 363 (0.21) (1.61)    (0.69*)   (0.53) N52S/C198R 125 1.80 82 1427  201 (2.09) (4.56)    (2.06)   (0.29) N52Y/N57Y/Y152C 126 0.08 56 377* 439 (0.09) (3.11)    (1.09*)   (0.64) N52Y/N57Y/ 127 ND 449 1192  ND H129P/C198R (ND) (24.94)   (1.72) (ND) N52H/L161P/C198R 128 0.18 343 643* 447 (0.21) (19.05)   (1.86*)    (0.65) N52S/T113E 129 1.51 54 451* 345 (1.76) (3.00)   (1.30*)    (0.50) S54A 130 1.62 48 386* 771 (1.88) (2.67)    (1.12*)   (1.12) N52D/S54P 368 1.50 38 476* 227 (1.74) (2.11)    (1.38*)   (0.33) N52K/L208P 132 1.91 291 1509  137 (2.22) (16.17)    (2.18)   (0.20) N52S/Y152H 133 0.85 68 2158  221 (0.99) (3.78)    (3.12)   (0.32) N52D/V151A 134 0.90 19 341* 450 (1.05) (1.06)    (0.99*)   (0.66) N52H/I143T 135 1.83 350 2216  112 (2.13) (19.44)    (3.20)   (0.16) N52S/L80P 136 0.09 22 192* 340 (0.10) (1.22)    (0.55*)   (0.49) F120S/Y152H/N201S 137 0.63 16 351* 712 (0.73) (0.89)   (1.01*)    (1.04) N52S/R75Q/L203P 138 1.71 12 1996  136 (1.99) (0.67)   (2.88)    (0.20) N52S/D158G 139 1.33 39 325* 277 (1.55) (2.17)   (0.94*)    (0.40) N52D/Q133H 140 1.53 104 365* 178 (1.78) (5.78)   (1.05*)    (0.26) WT ICOSL 32 0.86 18 692/346* 687 (1.00) (1.00)   (1.00)    (1.00) *Parental ratio calculated using 346 pg/ml IFN-gammafor WT ICOSL

Binding assays were repeated substantially as described above, exceptthat binding also was assessed against cells expressing full-lengthhuman CTLA4. ICOSL variant Fc fusion proteins also were further assessedin an anti-CD3 coimmobilization assay substantially as described above.The results confirmed identification of a number of ICOSL IgSF domainvariants that exhibited increased binding affinity for at least one, andin some cases more than one, cognate ligand. In addition, the resultsshowed that affinity modification of the variant molecules alsoexhibited improved activities in the coimmobilization assay.

Example 7 Additional Affinity Modified IgSF Domains

This examples describe the design, creation, and screening of additionalaffinity modified CD80 (B7-1), CD86 (B7-2) and NKp30 immunomodulatoryproteins, which are other components of the immune synapse (IS) thathave a demonstrated dual role in both immune activation and inhibition.These examples demonstrate that affinity modification of IgSF domainsyields proteins that can act to both increase and decrease immunologicalactivity. This work also describes the various combinations of thosedomains fused in pairs (i.e., stacked) with a variant affinity modifiedICOSL to form a Type II immunomodulatory protein to achieveimmunomodulatory activity.

Mutant DNA constructs of human CD80, CD86 and NKp30 IgSF domains fortranslation and expression as yeast display libraries were generatedsubstantially as described in Example 1. For libraries that targetspecific residues of target protein for complete or partialrandomization with degenerate codons, the coding DNA's for theextracellular domains (ECD) of human CD80 (SEQ ID NO:28), and NKp30 (SEQID NO:54) were ordered from Integrated DNA Technologies (Coralville,Iowa) as a set of overlapping oligonucleotides of up to 80 base pairs(bp) in length. Alternatively, residues were mutated by site-directedtargeted mutagenesis substantially as described in Example 1.Alternatively, random libraries were constructed to identify variants ofthe ECD of CD80 (SEQ ID NO:28), CD86 (SEQ ID NO: 29) and NKp30 (SEQ IDNO:54) substantially as described in Example 1.

The targeted and random library DNA was introduced into yeastsubstantially as described in Example 2 to generate yeast libraries. Thelibraries were used to select yeast expressing affinity modifiedvariants of CD80, CD86 and NKp30 substantially as described in Example3. Cells were processed to reduce non-binders and to enrich for CD80,CD86 or NKp30 variants with the ability to bind their exogenousrecombinant counter-structure proteins substantially as described inExample 3. For example, yeast displayed targeted or random CD80libraries were selected against each of CD28, CTL-4, and PD-L1,separately. This was then followed by two to three rounds of flowcytometry sorting using exogenous counter-structure protein staining toenrich the fraction of yeast cells that displays improved binders.Magnetic bead enrichment and selections by flow cytometry areessentially as described in Keith D. Miller,1 Noah B. Pefaur,2 andCheryl L. Baird1 Current Protocols in Cytometry 4.7.1-4.7.30, July 2008.

With CD80, CD86 and NKp30 libraries, target ligand proteins were sourcedfrom R&D Systems (USA) as follows: human rCD28.Fc (i.e., recombinantCD28-Fc fusion protein), rPDL1.Fc, rCTLA4.Fc, and rB7H6.Fc. Two-colorflow cytometry was performed substantially as described in Example 3.Yeast outputs from the flow cytometric sorts were assayed for higherspecific binding affinity. Sort output yeast were expanded andre-induced to express the particular IgSF affinity modified domainvariants they encode. This population then can be compared to theparental, wild-type yeast strain, or any other selected outputs, such asthe bead output yeast population, by flow cytometry.

In the case of NKp30 yeast variants selected for binding to B7-H6, theF2 sort outputs gave MFI values of 533 when stained with 16.6nMrB7H6.Fc, whereas the parental NKp30 strain MFI was measured at 90 whenstained with the same concentration of rB7H6.Fc (6-fold improvement).

Among the NKp30 variants that were identified, was a variant thatcontained mutations L30V/A60V/S64P/S86G with reference to positions inthe NKp30 extracellular domain corresponding to positions set forth inSEQ ID NO:54. Among the CD86 variants that were identified, was avariant that contained mutations Q35H/H9OL/Q102H with reference topositions in the CD86 extracellular domain corresponding to positionsset forth in SEQ ID NO:29. Among the CD80 variants that were identified,were variants set forth in Table 8 and described further below.

As with ICOSL, the MFIs of all F2 outputs described above when measuredwith the anti-HA tag antibody on FL1 did not increase and sometimes wentdown compared to wild-type strains, indicating that increased bindingwas not a function of increased expression of the selected variants onthe surface of yeast, and validated gating strategies of only selectingmid to low expressors with high ligand binding.

Exemplary selection outputs were reformatted as immunomodulatoryproteins containing an affinity modified (variant) extracellular domain(ECD) of CD80 fused to an Fc molecule (variant ECD-Fc fusion molecules)substantially as described in Example 4 and the Fc-fusion protein wasexpressed and purified substantially as described in Example 5.

Binding of exemplary CD80 Fc-fusion variants to cell-expressed counterstructures was then assessed substantially as described in Example 6. Toproduce cells expressing cognate binding partners, full-length mammaliansurface expression constructs for each of human CD28, CTLA4 and PD-L1were produced substantially as described in Example 6. Binding studiesand flow cytometry were carried out substantially as described inExample 6. In addition, the bioactivity of the Fc-fusion variant proteinwas characterized by either mixed lymphocyte reaction (MLR) or anti-CD3coimmobilization assay substantially as described in Example 6.

Results for the binding and activity studies for exemplary testedvariants are shown in Tables 8 and 9. In particular, Table 8 indicatesexemplary IgSF domain amino acid substitutions (replacements) in the ECDof CD80 selected in the screen for affinity-maturation against therespective cognate structure CD28. Table 9 indicates exemplary IgSFdomain amino acid substitutions (replacements) in the ECD of CD80selected in the screen for affinity-maturation against the respectivecognate structure PD-L1. As above, for each Table, the exemplary aminoacid substitutions are designated by amino acid position numbercorresponding to the respective reference unmodified ECD sequence asfollows. For example, the reference unmodified ECD sequence in Tables 8and 9 is the unmodified CD80 ECD sequence set forth in SEQ ID NO:28. Theamino acid position is indicated in the middle, with the correspondingunmodified (e.g. wild-type) amino acid listed before the number and theidentified variant amino acid substitution listed after the number.Column 2 sets forth the SEQ ID NO identifier for the variant ECD foreach variant ECD-Fc fusion molecule.

Also shown is the binding activity as measured by the Mean FluorescenceIntensity (MFI) value for binding of each variant Fc-fusion molecule tocells engineered to express the cognate counter structure ligand and theratio of the MFI compared to the binding of the corresponding unmodifiedECD-Fc fusion molecule not containing the amino acid substitution(s) tothe same cell-expressed counter structure ligand. The functionalactivity of the variant Fc-fusion molecules to modulate the activity ofT cells also is shown based on the calculated levels of IFN-gamma inculture supernatants (pg/ml) generated either i) with the indicatedvariant ECD-Fc fusion molecule coimmoblized with anti-CD3 or ii) withthe indicated variant ECD-Fc fusion molecule in an MLR assay. The Tablesalso depict the ratio of IFN-gamma produced by each variant ECD-Fccompared to the corresponding unmodified ECD-Fc in both functionalassays.

As shown, the selections resulted in the identification of a number ofCD80 IgSF domain variants that were affinity-modified to exhibitincreased binding for at least one, and in some cases more than one,cognate counter structure ligand. In addition, the results showed thataffinity modification of the variant molecules also exhibited improvedactivities to both increase and decrease immunological activitydepending on the format of the molecule. For example, coimmobilizationof the ligand likely provides a multivalent interaction with the cell tocluster or increase the avidity to favor agonist activity and increase Tcell activation compared to the unmodified (e.g. wildtype) ECD-Fcmolecule not containing the amino acid replacement(s). However, when themolecule is provided as a bivalent Fc molecule in solution, the sameIgSF domain variants exhibited an antagonist activity to decrease T cellactivation compared to the unmodified (e.g. wildtype) ECD-Fv moleculenot containing the amino acid replacement(s).

TABLE 8 CD80 variants selected against CD28. Molecule sequences, bindingdata, and costimulatory bioactivity data. Coimmobil- ization withBinding anti-CD3 MLR IFN- CD28 CTLA-4 PD-L1 IFN-gamma gamma levels SEQMFI MFI MFI pg/ml pg/ml ID NO (parental (parental (parental (parental(parental CD80 mutation(s) (ECD) ratio) ratio) ratio) ratio) ratio)L70Q/A91G/N144D 508 125 283 6 93 716 (1.31) (1.36) (0.08) (1.12) (0.83)L70Q/A91G/T130A 56 96 234 7 99 752 (1.01) (1.13) (0.10) (1.19) (0.87)L70Q/A91G/I118A/ 59 123 226 7 86 741 T120S/T130A/K169E (1.29) (1.09)(0.10) (1.03) (0.86) V4M/L70Q/A91G/I118V/ 510 89 263 6 139 991T120S/T130A/K169E (0.94) (1.26) (0.09) (1.67) (1.14) L70Q/A91G/I118V/ 59106 263 6 104 741 T120S/T130A/K169E (1.12) (1.26) (0.09) (1.25) (0.86)V20L/L70Q/A91S/ 513 105 200 9 195 710 I118V/T120S/T130A (1.11) (0.96)(0.13) (2.34) (0.82) S44P/L70Q/A91G/ 61 88 134 5 142 854 T130A (0.92)(0.64) (0.07) (1.71) (0.99) L70Q/A91G/E117G/ 514 120 193 6 98 736I118V/T120S/T130A (1.27) (0.93) (0.08) (1.05) (0.85) A91G/I118V/T120S/515 84 231 44 276 714 T130A (0.89) (1.11) (0.62) (3.33) (0.82)L70R/A91G/I118V/ 516 125 227 6 105 702 T120S/T130A/T199S (1.32) (1.09)(0.09) (1.26) (0.81) L70Q/E81A/A91G/ 517 140 185 18 98 112I118V/T120S/I127T/ (1.48) (0.89) (0.25) (1.18) (0.89) T130AL70Q/Y87N/A91G/ 66 108 181 6 136 769 T130A (1.13) (0.87) (0.08) (1.63)(0.89) T28S/L70Q/A91G/ 518 32 65 6 120 834 I118V/E95K/T120S/ (0.34)(0.31) (0.08) (1.44) (0.96) I126V/T130A/K169E N63S/L70Q/A91G/ 519 124165 6 116 705 S114T/I118V/T120S/ (1.30) (0.79) (0.08) (1.39) (0.81)T130A K36E/I67T/L70Q/ 520 8 21 5 53 852 A91G/I118V/T120S/ (0.09) (0.10)(0.08) (0.63) (0.98) T130A/N152T E52G/L70Q/A91G/ 521 113 245 6 94 874D107N/I118V/T120S/ (1.19) (1.18) (0.08) (1.13) (1.01) T130A K169EK37E/F59S/L70Q/ 522 20 74 6 109 863 A91G/I118V/T120S/ (0.21) (0.36)(0.08) (1.31) (1.00) T130A/K185E A91G/S103P 72 39 56 9 124 670 (0.41)(0.27) (0.13) (1.49) (0.77) K89E/T130A 73 90 148 75 204 761 (0.95)(0.71) (1.07) (2.45) (0.88) A91G 74 96 200 85 220 877 (1.01) (0.96)(1.21) (2.65) (1.01) D60V/A91G/I118V/ 523 111 222 12 120 744T120S/T130A/K169E (1.17) (1.07) (0.18) (1.44) (0.86) K54M/L70Q/A91G/ 52468 131 5 152 685 Y164H (0.71) (0.63) (0.08) (1.83) (0.79)M38T/L70Q/E77G/ 525 61 102 5 119 796 A91G/I118V/T120S/ (0.64) (0.49)(0.07) (1.43) (0.92) T130A/N152T R29H/E52G/L70R/ 78 100 119 5 200 740E88G/A91G/T130A (1.05) (0.57) (0.08) (2.41) (0.85) Y31H/T41G/M43L/ 52685 85 6 288 782 L70Q/A91G/I118V/ (0.89) (0.41) (0.08) (3.47) (0.90)T120S/I126V/T130A V68A/T110A 80 103 233 48 163 861 (1.08) (1.12) (0.68)(1.96) (0.99) L65H/D90G/T110A/ 527 33 121 11 129 758 F116L (0.35) (0.58)(0.15) (1.55) (0.88) R29H/E52G/D90N/ 82 66 141 11 124 800I118V/T120S/T130A (0.69) (0.68) (0.15) (1.49) (0.92) A91G/L102S 83 6 6 575 698 (0.06) (0.03) (0.08) (0.90) (0.81) I67T/L70Q/A91G/ 530 98 160 51751 794 I118V T120S (1.03) (0.77) (0.08) (21.1) (0.92) L70Q/A91G/T110A/531 8 14 5 77 656 I118V/T120S/T130A (0.09) (0.07) (0.07) (0.93) (0.76)M38V/T41D/M43I/ 532 5 8 8 82 671 W50G/D76G/V83A/ (0.06) (0.04) (0.11)(0.99) (0.78) K89E/I118V/T120S/ I126V/T130A V22A/L70Q/S121P 87 5 7 5 105976 (0.06) (0.04) (0.07) (1.27) (1.13) A12V/S15F/Y31H/ 533 6 6 5 104 711M38L/T41G/M43L/ (0.06) (0.03) (0.08) (1.25) (0.82) D90N/T130A/P137L/N149D N152T I67F/L70R/E88G/ 534 5 6 6 62 1003 A91G/I118V/T120S/ (0.05)(0.03) (0.08) (0.74) (1.16) T130A E24G/L25P/L70Q/ 535 26 38 8 101 969A91G/I118V/T120S/ (0.27) (0.18) (0.11) (1.21) (1.12) N152TA91G/F92L/F108L/ 536 50 128 16 59 665 I118V/T120S (0.53) (0.61) (0.11)(0.71) (0.77) WT CD80 28 95 208 70 83 866 (1.00) (1.00) (1.00) (1.00)(1.00)

TABLE 9 CD80 variants selected against PD-L1. Molecule sequences,binding data, and costimulatory bioactivity data. Coimmobil- izationwith Binding anti-CD3 MLR IFN- CD28 CTLA-4 PD-L1 IFN-gamma gamma levelsSEQ MFI MFI MFI pg/ml pg/ml ID NO (parental (parental (parental(parental (parental CD80 mutation(s) (ECD) ratio) ratio) ratio) ratio)ratio) R29D/Y31L/Q33H/ 92 1071 1089 37245 387 5028 K36G/M38I/T41A/(0.08) (0.02) (2.09) (0.76) (0.26) M43R/M47T/E81V/ L85R/K89N/A91T/F92P/K93V/R94L/ I118T/N149S R29D/Y31L/Q33H/ 93 1065 956 30713 400 7943K36G/M38I/T41A/ (0.08) (0.02) (1.72) (0.79) (0.41) M43R/M47T/E81V/L85R/K89N/A91T/ F92P/K93V/R94L/ N144S/N149S R29D/Y31L/Q33H/ 94 926 95447072 464 17387 K36G/M38I/T41A/ (0.07) (0.02) (2.64) (0.91) (0.91)M42T/M43R/M47T/ E81V/L85R/K89N/ A91T/F92P/K93V/ R94L/L148S/N149SE24G/R29D/Y31L/ 95 1074 1022 1121 406 13146 Q33H/K36G/M38I/ (0.08)(0.02) (0.06) (0.80) (0.69) T41A/M43R/M47T/ F59L/E81V/L85R/K89N/A91T/F92P/ K93V/R94L/H96R R29D/Y31L/Q33H/ 96 1018 974 25434 40524029 K36G/M38I/T41A/ (0.08) (0.02) (1.43) (0.80) (1.25) M43R/M47T/E81V/L85R/K89N/A91T/ F92P/K93V/R94L/ N149S R29V/M43Q/E81R/ 97 1029 996 1575342 11695 L85I/K89R/D90L/ (0.08) (0.02) (0.09) (0.67) (0.61)A91E/F92N/K93Q/ R94G T41I/A91G 98 17890 50624 12562 433 26052 (1.35)(1.01) (0.70) (0.85) (1.36) E88D/K89R/D90K/ 537 41687 49429 20140 7736345 A91G/F92Y/K93R/ (3.15) (0.99) (1.13) (1.52) (0.33) N122S/N178SE88D/K89R/D90K/ 538 51663 72214 26405 1125 9356 A91G/F92Y/K93R (3.91)(1.44) (1.48) (2.21) (0.49) K36G/K37Q/M38I/ 539 1298 1271 3126 507 3095L40M/F59L/E81V/ (0.10) (0.03) (0.18) (1.00) (0.16) L85R/K89N/A91T/F92P/K93V/R94L/ E99G/T130A/N149S AE88D/K89R/D90K/ 102 31535 50868 29077944 5922 A91G/F92Y/K93R (2.38) (1.02) (1.63) (1.85) (0.31)K36G/K37Q/M38I/ 103 1170 1405 959 427 811 L40M (0.09) (0.03) (0.05)(0.84) (0.04) K36G/L40M 540 29766 58889 20143 699 30558 (2.25) (1.18)(1.13) (1.37) (1.59) WTCD80 28 13224 50101 17846 509 19211 (1.00) (1.00)(1.00) (1.00) (1.00)

Example 8 Generation and Assessment of Stacked Molecules ContainingDifferent Affinity-Modified Domains

This Example describes further immunomodulatory proteins that weregenerated as stack constructs containing at least two different affinitymodified domains from identified variant ICOSL polypeptides and one moreadditional variant CD80, CD86, ICOSL, and NKp30 molecules linkedtogether and fused to an Fc.

Selected variant molecules described above that were affinity-modifiedfor one or more counter structure ligand were used to generate “stack”molecule (i.e., Type II immunomodulatory protein) containing two or moreaffinity-modified IgSF domains. Stack constructs were obtained asgeneblocks (Integrated DNA Technologies, Coralville, Iowa) that encodethe stack in a format that enables its fusion to Fc by standard Gibsonassembly using a Gibson assembly kit (New England Biolabs).

The encoding nucleic acid molecule of all stacks was generated to encodea protein designed as follows: Signal peptide, followed by the firstvariant IgV of interest, followed by a 15 amino acid linker which iscomposed of three GGGGS(G4S) motifs (SEQ ID NO:228), followed by thesecond IgV of interest, followed by two GGGGS linkers (SEQ ID NO: 229)followed by three alanines (AAA), followed by a human IgG1 Fc asdescribed above. To maximize the chance for correct folding of the IgVdomains in each stack, the first IgV was preceded by all residues thatnormally occur in the wild-type protein between this IgV and the signalpeptide (leading sequence). Similarly, the first IgV was followed by allresidues that normally connect it in the wild-type protein to either thenext Ig domain (typically an IgC domain) or if such a second IgV domainis absent, the residues that connect it to the transmembrane domain(trailing sequence). The same design principle was applied to the secondIgV domain except that when both IgV domains were derived from sameparental protein (e.g. a ICOSL IgV stacked with another ICOSL IgV), thelinker between both was not duplicated.

Table 10 sets forth the design for exemplary stacked constructs. Theexemplary stack molecules shown in Table 10 contains the Ig domains(e.g. IgV domain) as indicated and additionally trailing sequences asdescribed above. In the Table, the following components are present inorder: signal peptide (SP; SEQ ID NO:225), Ig domain 1 (e.g. Igl),trailing sequence 1 (TS1), linker 1 (LR1; SEQ ID NO:228), Ig domain 2(Ig2), trailing sequence 2 (TS2), linker 2 (LR2; SEQ ID NO:230) and Fcdomain (SEQ ID NO:226 containing C5S/R77C/N82G/V87C amino acidsubstitution). In some cases, a leading sequence l(LS1) is presentbetween the signal peptide and IgV1 and in some cases a leading sequence2 (LS2) is present between the linker and IgV2.

TABLE 10 Amino acid sequence (SEQ ID NO) of components of exemplarystacked constructs First domain Second domain SP LS1 Ig1 TS1 LR1 LS2 Ig2TS2 LR2 Fc Domain 1: NKp30 + − 214 235 + − 196 233 + + WT Domain 2:ICOSL WT Domain 1: NKp30 + − 215 235 + − 212 233 + + L30V/A60V/S64P/S86G Domain 2: ICOSL N52S/N57Y/H94D/ L96F/L98F/Q100R Domain 1: NKp30 + −215 235 + − 199 233 + + L30V/A60V/S64P/ S86G) Domain 2: ICOSL N52DDomain 1: NKp30 + − 215 235 + − 201 233 + + L30V/A60V/S64P/ S86G Domain2: ICOSL N52H/N57Y/Q100P Domain 1: ICOSL + − 196 233 + − 214 235 + + WTDomain 2: Nkp30 WT Domain 1: ICOSL + − 199 233 + − 215 235 + + N52DDomain 2: NKp30 L30V/A60V/S64P/ S86G Domain 1: ICOSL + − 201 233 + − 215235 + + N52H/N57Y/Q100P Domain 2: NKp30 L30V/A60V/S64P/ S86G Domain 1:CD80 + − 152 471 + − 196 233 + + WT Domain 2: ICOSL WT Domain 1: CD80 +− 189 471 + − 213 233 + + E88D/K89R/D90K/ A91G/F92Y/K93R Domain 2: ICOSLN52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/F120S Domain 1: CD80 + − 193471 + − 213 233 + + A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 2:ICOSL N52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/F120S Domain 1: CD80 + −193 471 + − 199 233 + + A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 2:ICOSL N52D Domain 1: CD80 + − 189 471 + − 201 233 + + E88D/K89R/D90K/A91G/F92Y/K93R Domain 2: ICOSL N52H/N57Y/Q100P Domain 1: CD80 + − 193471 + − 201 233 + + A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 2:ICOSL N52H/N57Y/Q100P Domain 1: ICOSL + − 196 233 + − 152 471 + + WTDomain 2: CD80 WT Domain 1: ICOSL + − 213 233 + − 189 471 + +N52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/F120S Domain 2: CD80E88D/K89R/D90K/ A91G/F92Y/K93R Domain 1: ICOSL + − 213 233 + − 193471 + + N52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/F120S Domain 2: CD80A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 1: ICOSL + − 199 233 + −189 471 + + N52D Domain 2: CD80 E88D/K89R/D90K/ A91G/F92Y/K93R Domain 1:ICOSL + − 199 233 + − 193 471 + + N52D Domain 2: CD80 A12T/H18L/M43V/F59L/E77K/P109S/ I118T Domain 1: ICOSL + − 201 233 + − 189 471 + +N52H/N57Y/Q100P Domain 2: CD80 E88D/K89R/D90K/ A91G/F92Y/K93R Domain 1:ICOSL + − 201 233 + − 193 471 + + N52H/N57Y/Q100P Domain 2: CD80A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 1: CD86 + 236 220 237 + −196 233 + + WT Domain 2: ICOSL WT Domain 1: CD80 + − 192 471 + − 213233 + + R29H/Y31H/T41G/ Y87N/E88G/K89E/ D90N/A91G/P109S Domain 2: ICOSLN52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/F120S Domain 1: CD80 + − 175471 + − 213 233 + + I67T/L70Q/A91G/ T120S Domain 2: ICOSLN52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/F120S Domain 1: CD80 + − 192471 + − 199 233 + + R29H/Y31H/T41G/ Y87N/E88G/K89E/ D90N/A91G/P109SDomain 2: ICOSL N52D Domain 1: CD80 + − 175 471 + − 199 233 + +I67T/L70Q/A91G/ T120S Domain 2: ICOSL N52D Domain 1: CD80 + − 192 471 +− 201 233 + + R29H/Y31H/T41G/ Y87N/E88G/K89E/ D90N/A91G/P109S Domain 2:ICOSL N52H/N57Y/Q100P Domain 1: CD80 + − 175 471 + − 201 233 + +I67T/L70Q/A91G/ T120S Domain 2: ICOSL N52H/N57Y/Q100P Domain 1: CD86 +236 221 237 + − 213 233 + + Q35H/H90L/Q102H Domain 2: ICOSLN52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/F120S Domain 1: CD86 + 236 221237 + − 199 233 + + Q35H/H90L/Q102H Domain 2: ICOSL N52D Domain 1:CD86 + 236 221 237 + − 201 233 + + Q35H/H90L/Q102H Domain 2: ICOSLN52H/N57Y/Q100P Domain 1: ICOSL + − 196 233 + 236 220 237 + + WT Domain2: CD86 WT Domain 1: ICOSL + − 213 233 + − 192 471 + + N52S/N57Y/H94D/L96F/L98F/Q100R/ G103E/F120S Domain 2: CD80 R29H/Y31H/T41G/Y87N/E88G/K89E/ D90N/A91G/P109S Domain 1: ICOSL + − 213 233 + − 175471 + + N52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/F120S Domain 2: CD80I67T/L70Q/A91G/ T120S Domain 1: ICOSL + − 199 233 + − 192 471 + + N52DDomain 2: CD80 R29H/Y31H/T41G/ Y87N/E88G/K89E/ D90N/A91G/P109S Domain 1:ICOSL + − 199 233 + − 175 471 + + N52D Domain 2: CD80 I67T/L70Q/A91G/T120S Domain 1: ICOSL + − 201 233 + − 192 471 + + N52H/N57Y/Q100P Domain2: CD80 R29H/Y31H/T41G/ Y87N/E88G/K89E/ D90N/A91G/P109S Domain 1:ICOSL + − 213 233 + 236 221 237 + + N52S/N57Y/H94D/ L96F/L98F/Q100R/G103E/F120S Domain 2: CD86 Q35H/H90L/Q102H Domain 1: ICOSL + − 199 233 +236 221 237 + + N52D Domain 2: CD86 Q35H/H90L/Q102H Domain 1: ICOSL + −201 233 + 236 221 237 + + N52H/N57Y/Q100P Domain 2: CD86 Q35H/H90L/Q102H

High throughput expression and purification of the variantIgV-stacked-Fc fusion molecules containing various combinations ofvariant IgV domains from CD80, CD86, ICOSL or Nkp30 containing at leastone affinity-modified IgV domain were generated substantially asdescribed in Example 5. Binding of the variant IgV-stacked-Fc fusionmolecules to respective counter structures and functional activity byanti-CD3 coimmobilization assay also were assessed substantially asdescribed in Example 6. For example, costimulatory bioactivity of thestacked IgSF Fc fusion proteins was determined in a similar immobilizedanti-CD3 assay as above. In this case, 4 nM of anti-CD3 (OKT3,Biolegend, USA) was coimmobilized with 4 nM to 120 nM of human rB7-H6.Fc(R&D Systems, USA) or human rPD-L1.Fc (R&D Systems, USA) overnight ontissue-culture treated 96-well plates (Corning, USA). The following dayunbound protein was washed off with PBS and 100,000 purified pan T cellswere added to each well in 100 μl Ex-Vivo 15 media (Lonza, Switzerland).The stacked IgSF domains were subsequently added at concentrationsranging from 8 nM to 40 nM in a volume of 100 μl for 200 μl volumetotal. Cells were cultured 3 days before harvesting culture supernatantsand measuring human IFN-gamma levels with Duoset ELISA kit (R&D Systems,USA) as mentioned above.

The results are set forth in Tables 11-13. Specifically, Table 11 setsforth binding and functional activity results for variant IgV-stacked-Fcfusion molecules containing an NKp30 IgV domain and an ICOSL IgV domain.Table 12 and 13 sets forth binding and functional activity results forvariant IgV-stacked-Fc fusion molecules containing a variant ICOSL IgVdomain and a variant CD80 IgV or CD86 IgV domain.

For each of Tables 11-13, Column 1 indicates the structural organizationand orientation of the stacked, affinity modified or wild-type (WT)domains beginning with the amino terminal (N terminal) domain, followedby the middle WT or affinity modified domain located before the Cterminal human IgG1 Fc domains. Column 2 sets forth the SEQ ID NOidentifier for the sequence of each IgV domain contained in a respective“stack” molecule. Column 3 shows the binding partners which theindicated affinity modified stacked domains from column 1 were selectedagainst.

Also shown is the binding activity as measured by the Mean FluorescenceIntensity (MFI) value for binding of each stack molecule to cellstransfected to express various counter structure ligands and the ratioof the MFI compared to the binding of the corresponding stack moleculecontaining unmodified IgV domains not containing the amino acidsubstitution(s) to the same cell-expressed counter structure ligand. Thefunctional activity of the variant stack molecules to modulate theactivity of T cells also is shown based on the calculated levels ofIFN-gamma in culture supernatants (pg/ml) generated with the indicatedvariant stack molecule in solution and the appropriate ligandcoimmoblized with anti-CD3 as described in Example 6. The Table alsodepicts the ratio of IFN-gamma produced by each variant stack moleculecompared to the corresponding unmodified stack molecule in thecoimmobilization assay.

As shown, the results showed that it was possible to generate stackmolecules containing at least one variant IgSF domains that exhibitedaffinity-modified activity of increased binding for at least one cognatecounter structure ligand compared to a corresponding stack moleculecontaining the respective unmodified (e.g. wild-type) IgV domain. Insome cases, the stack molecule, either from one or a combination of bothvariant IgSF domains in the molecule, exhibited increased binding formore than one cognate counter structure ligand. The results also showedthat the order of the IgV domains in the stacked molecules could, insome cases, alter the degree of improved binding activity. In somecases, functional T cell activity also was altered when assessed in thetargeted coimmobilization assay.

TABLE 11 Stacked variant IgV Fc fusion proteins containing an NKp30 IgVdomain and an ICOSL IgV domain Anti-CD3 coimmobil- Binding Activityization assay Counter B7H6 MFI ICOS MFI CD28 MFI pg/ml IFN- DomainStructure SEQ ID structure (WT (WT (WT gamma (WT N terminal to Cterminal: NO (Ig selected parental parental parental parental IFN-domain 1/domain 2/Fc domain) against MFI ratio) MFI ratio) MFI ratio)gamma ratio) Domain 1: NKp30 WT 214 — 64538 26235 6337 235 Domain 2:ICOSL WT 196 (1.00) (1.00) (1.00) (1.00) Domain 1: NKp30 215 B7-H6 5968412762 9775 214 L30V/A60V/S64P/S86G (0.92) (0.49) (1.54) (0.91) Domain 2:ICOSL 212 ICOS-CD28 N52S N57Y H94D L96F L98F Q100R Domain 1: NKp30 215B7-H6 65470 30272 9505 219 L30V/A60V/S64P/S86G (1.01) (1.15) (1.50)(0.93) Domain 2: ICOSL 199 ICOS-CD28 N52D Domain 1: NKp30 215 B7-H638153 27903 11300 189 L30V/A60V/S64P/S86G (0.59) (1.06) (1.78) (0.80)Domain 2: ICOSL 201 ICOS-CD28 N52H N57Y Q100P Domain 1: ICOSL WT 196117853 70320 7916 231 Domain 2: Nkp30 WT 214 — (1.0) (1.0) (1.0) (1.0)Domain 1: ICOSL 199 ICOS-CD28 100396 83912 20778 228 N52D (0.85) (1.19)(2.62) (0.98) Domain 2: NKp30 215 B7-H6 L30V/A60V/S64P/S86G Domain 1:ICOSL 201 ICOS-CD28 82792 68874 72269 561 N52H/N57Y/Q100P (0.70) (0.98)(9.12) (2.43) Domain 2: NKp30 215 B7-H6 L30V/A60V/S64P/S86G

TABLE 12 Stacked variant IgV Fc fusion proteins containing a CD80 IgVdomain and a ICOSL IgV domain Anti-CD3 coimmobil- Binding Activityization assay Counter CD28 MFI PD-L1 MFI ICOS MFI pg/ml IFN- DomainStructure SEQ ID structure (WT (WT (WT gamma (WT N terminal to Cterminal: NO (Ig selected parental parental parental parental IFN-domain 1/domain 2/Fc domain) against MFI ratio) MFI ratio) MFI ratio)gamma ratio) Domain 1: CD80 WT 152 1230 2657 11122 69 Domain 2: ICOSL WT196 (1.00) (1.00) (1.00) (1.00) Domain 1: CD80 189 PD-L1 3383 4515 515890 E88D/K89R/D90K/A91G/ (2.75) (1.70) (0.46) (1.30) F92Y/K93R Domain 2:ICOSL 213 ICOS/CD28 N52S/N57Y/H94D/L96F/ L98F/Q100R/G103E/ F120S Domain1: CD80 193 PD-L1 2230 2148 3860 112 A12T/H18L/M43V/F59L/ (1.81) (0.81)(0.35) (1.62) E77K/P109S/I118T Domain 2: ICOSL 213 ICOS/CD28N52S/N57Y/H94D/L96F/ L98F/Q100R/G103E/ F120S Domain 1: CD80 193 PD-L15665 6446 15730 126 A12T/H18L/M43V/F59L/ (4.61) (2.43) (1.41) (1.83)E77K/P109S/I118T Domain 2: ICOSL 199 ICOS/CD28 N52D Domain 1: CD80 189PD-L1 6260 4543 11995 269 E88D/K89R/D90K/A91G/ (5.09) (1.71) (1.08)(3.90) F92Y/K93R Domain 2: ICOSL 201 ICOS/CD28 N52H/N57Y/Q100P Domain 1:CD80 193 PD-L1 3359 3874 8541 97 A12T/H18L/M43V/F59L/ (2.73) (1.46)(0.77) (1.41) E77K/P109S/I118T Domain 2: ICOSL 201 ICOS/CD28N52H/N57Y/Q100P Domain 1: ICOSL WT 196 3000 2966 14366 101 Domain 2:CD80 WT 152 (1.00) (1.00) (1.00) (1.00) Domain 1: ICOSL 213 ICOS/CD283634 4893 6403 123 N52S/N57Y/H94D/L96F/ (1.21) (1.65) (0.45) (1.22)L98F/Q100R/G103E/ F120S Domain 2: CD80 189 PD-L1 E88D/K89R/D90K/A91G/F92Y/K93R Domain 1: ICOSL 213 ICOS/CD28 1095 5929 7923 127N52S/N57Y/H94D/L96F/ (0.37) (2.0) (0.55) (1.26) L98F/Q100R/G103E/ F120SDomain 2: CD80 193 PD-L1 A12T/H18L/M43V/F59L/ E77K/P109S/I118T Domain 1:ICOSL 199 ICOS/CD28 2023 5093 16987 125 N52D (0.67) (1.72) (1.18) (1.24)Domain 2: CD80 189 PD-L1 E88D/K89R/D90K/A91G/ F92Y/K93R Domain 1: ICOSL199 ICOS/CD28 3441 3414 20889 165 N52D (1.15) (1.15) (1.45) (1.63)Domain 2: CD80 193 PD-L1 A12T/H18L/M43V/F59L/ E77K/P109S/I118T Domain 1:ICOSL 201 ICOS/CD28 7835 6634 20779 95 N52H/N57Y/Q100P (2.61) (2.24)(1.45) (0.94) Domain 2: CD80 189 PD-L1 E88D/K89R/D90K/A91G/ F92Y/K93RDomain 1: ICOSL 201 ICOS/CD28 8472 3789 13974 106 N52H/N57Y/Q100P (2.82)(1.28) (0.97) (1.05) Domain 2: CD80 193 PD-L1 A12T/H18L/M43V/F59L/E77K/P109S/I118T

TABLE 13 Stacked variant IgV Fc fusion proteins containing a CD80 orCD86 IgV domain and an ICOSL IgV domain SEQ ID Counter Binding ActivityFunctional Domain Structure NO structure PD-L1 MFI CTLA-4 MFI Activity Nterminal to C terminal: (Ig selected (WT parental (WT parental MLRIFN-gamma domain 1/domain 2/Fc domain) against MFI ratio) MFI ratio)pg/ml Domain 1: CD80 WT 152 1230 11122 1756 Domain 2: ICOSL WT 196(1.00) (1.00) (1.00) Domain 1: CD86 WT 220 29343 55193 6305 Domain 2:ICOSL WT 196 (1.00) (1.00) (1.00) Domain 1: CD80 192 CD28 2280 3181 2281R29H/Y31H/T41G/Y87N/ (1.85) (0.29) (1.30) E88G/K89E/D90N/A91G/ P109SDomain 2: ICOSL 213 ICOS/CD28 N52S/N57Y/H94D/L96F/L98F/Q100R/G103E/F120S Domain 1: CD80 175 CD28 2309 26982 1561I67T/L70Q/A91G/T120S (1.88) (2.43) (0.89) Domain 2: ICOSL 213 ICOS/CD28N52S/N57Y/H94D/L96F/ L98F/Q100R/G103E/F120S Domain 1: CD80 192 CD28 428522744 1612 R29H/Y31H/T41G/Y87N/ (3.48) (2.04) (0.92)E88G/K89E/D90N/A91G/ P109S Domain 2: ICOSL 199 ICOS/CD28 N52D Domain 1:CD80 175 CD28 3024 16916 3857 I67T/L70Q/A91G/T120S (2.46) (1.52) (2.20)Domain 2: ICOSL 199 ICOS/CD28 N52D Domain 1: CD80 192 CD28 6503 72406886 R29H/Y31H/T41G/Y87N/ (5.29) (0.65) (3.92) E88G/K89E/D90N/A91G/P109S Domain 2: ICOSL 201 ICOS/CD28 N52H/N57Y/Q100P Domain 1: CD80 175CD28 3110 4848 3393 I67T/L70Q/A91G/T120S (2.53) (0.44) (1.93) Domain 2:ICOSL 201 ICOS/CD28 N52H/N57Y/Q100P Domain 1: CD86 221 CD28 11662 21165880 Q35H/H90L/Q102H (0.40) (0.38) (0.14) Domain 2: ICOSL 213 ICOS/CD28N52S/N57Y/H94D/L96F/ L98F/Q100R/G103E/F120S Domain 1: CD86 221 CD2824230 73287 1110 Q35H/H90L/Q102H (0.83) (1.33) (0.18) Domain 2: ICOSL199 ICOS/CD28 N52D Domain 1: CD86 221 CD28 1962 1630 587 Q35H/H90L/Q102H(0.07) (0.03) (0.09) Domain 2: ICOSL 201 ICOS/CD28 N52H/N57Y/Q100PDomain 1: ICOSL WT 196 3000 14366 4113 Domain 2: CD80 WT 152 (1.00)(1.00) (1.00) Domain 1: ICOSL WT 196 18005 53602 18393 Domain 2: CD86 WT220 (1.00) (1.00) (1.00) Domain 1: ICOSL 213 ICOS/CD28 10426 51286 18680N52S/N57Y/H94D/L96F/ (3.48) (3.57) (4.54) L98F/Q100R/G103E/F120S Domain2: CD80 192 CD28 R29H/Y31H/T41G/Y87N/ E88G/K89E/D90N/A91G/ P109S Domain1: ICOSL 213 ICOS/CD28 17751 29790 10637 N52S/N57Y/H94D/L96F/ (5.92)(2.07) (2.59) L98F/Q100R/G103E/F120S Domain 2: CD80 175 CD28I67T/L70Q/A91G/T120S Domain 1: ICOSL 199 ICOS/CD28 2788 25870 6205 N52D(0.93) (1.80) (1.51) Domain 2: CD80 192 CD28 R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/ P109S Domain 1: ICOSL 199 ICOS/CD28 2522 13569 5447N52D (0.84) (0.94) (1.32) Domain 2: CD80 175 CD28 I67T/L70Q/A91G/T120SDomain 1: ICOSL 201 ICOS/CD28 9701 9187 5690 N52H/N57Y/Q100P (3.23)(0.64) (1.38) Domain 2: CD80 192 CD28 R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/ P109S Domain 1: ICOSL 213 ICOS/CD28 27050 212578131 N52S/N57Y/H94D/L96F/ (1.50) (0.40) (0.44) L98F/Q100R/G103E/F120SDomain 2: CD86 221 CD28 Q35H/H90L/Q102H Domain 1: ICOSL 199 ICOS/CD2834803 80210 6747 N52D (1.93) (1.50) (0.37) Domain 2: CD86 221 CD28Q35H/H90L/Q102H Domain 1: ICOSL 201 ICOS/CD28 5948 4268 26219N52H/N57Y/Q100P (0.33) (0.08) (1.43) Domain 2: CD86 221 CD28Q35H/H90L/Q102H

Example 9 Generation and Assessment of Engineered Cells Expressing aTransmembrane Immunomodulatory Protein

Engineered T cells were generated in which a transmembraneimmunomodulatory protein (TIP) containing an extracellular domain (ECD)containing either a variant CD80 as described above or an ICOSLaffinity-modified IgSF domain was co-expressed with a chimeric antigenreceptor (CAR). The TIP also contained a transmembrane domain and acytoplasmic domain of the corresponding wild-type CD80 or ICOSLtransmembrane protein sequence. The immunomodulatory activity of theengineered cells was compared to cells that only expressed the CAR orcells that co-expressed the corresponding wild-type CD80 or ICOSLtransmembrane protein with the CAR.

The exemplary CD80-TIP was a variant CD80 having an affinity-modifiedIgSF domain containing amino acid mutations in the IgV and IgC domainscorresponding to 167T/L70Q/A91G/T120S with reference to positions in theCD80 extracellular domain set forth in SEQ ID NO:28 and a transmembraneand cytoplasmic domain corresponding to residues 243-288 of SEQ ID NO:l.The amino acid sequence of the exemplary CD80-TIP is set forth in SEQ IDNO:241 and is encoded by the sequence of nucleotides set forth in SEQ IDNO:242. The corresponding wild-type CD80 transmembrane protein had thesequence of amino acids set forth as amino acid residues 35-288 of SEQID NO:1 and encoded by the sequence of amino acids set forth in SEQ IDNO: 251.

The exemplary ICOSL-TIP was a variant ICOSL having an affinity-modifiedIgSF domain containing amino acid mutations in the IgV domaincorresponding to N52H/I143T with reference to positions in the ICOSLextracellular domain set forth in SEQ ID NO:32 and a transmembrane andcytoplasmic domain corresponding to residues 257-302 of SEQ ID NO:5. Theamino acid sequence of the exemplary ICOSL-TIP is set forth in SEQ IDNO:243 and is encoded by the sequence of nucleotides set forth in SEQ IDNO:244. The corresponding wild-type ICOSL transmembrane protein had thesequence of amino acids set forth as amino acid residues 19-302 of SEQID NO:5 and encoded by the sequence of amino acids set forth in SEQ IDNO: 252.

The TIP containing the affinity-modified domain or the wild-typetransmembrane protein containing a corresponding non-affinity modifiedIgSF domain were co-expressed in T cells with a 1^(st) generationchimeric antigen receptor (CAR) containing a CD3zeta intracellularsignaling domain. The 1^(st) generation CAR included an scFv specificfor CD19 (SEQ ID NO:245), a hinge and transmembrane domain derived fromCD8 (SEQ ID NO:246) and an intracellular signaling domain derived fromCD3zeta (set forth in SEQ ID NO:47). The nucleotide sequence encodingthe CD19 scFv-CD3zeta CAR is set forth in SEQ ID NO:248 and the aminoacid sequence of the CD19 scFv-CD3zeta CAR is set forth in SEQ IDNO:479.

Nucleic acid molecules encoding the CAR alone or also encoding one ofthe exemplary TIPs or wild-type transmembrane proteins separated fromthe CAR by a self-cleaving T2A sequence (SEQ ID NO:250 and encoded bythe sequence of nucleotides set forth in SEQ ID NO:249) were generated.Exemplary constructs contained nucleic acid sequences set forth in Table14. As a control, a nucleic acid construct encoding a 2^(nd) generationCAR additionally containing a CD28 costimulatory domain also wasgenerated (CD19 scFv-CD28-CD3zeta).

TABLE 14 Nucleic Acid Constructs CAR (SEQ T2A Linker TIP ID NO) (SEQ IDNO) (SEQ ID NO) CD19 scFv - CD3zeta + − − (248) CD19 scFv - CD3zeta -T2A - + + Wildtype CD80 B7-1 (248) (249) (251) CD19 scFv - CD3zeta -T2A - + + CD80 TIP B7-1_TIP (248) (249) (242) CD19 scFv - CD3zeta -T2A - + + Wildtype ICOSL (248) (249) ICOSL (252) CD19 scFv - CD3zeta -T2A - + + ICOSL TIP ICOSL_TIP (248) (249) (244)

The nucleic acid molecules were individually cloned into a lentiviralvector, which was used to transduce T cells isolated from human PBMCsamples obtained from three different healthy donors. Lentivirusparticles containing the nucleic acid sequences were produced afterco-transfection of HEK293 cells with the vectors and lentiviruspackaging constructs. The lentivirus particles were collected from theculture medium by ultracentrifugation and titered by qRT-PCR. Humanperipheral blood mononuclear cells (PBMC) were isolated from threenormal blood donors using density sedimentation. The PBMC were culturedovernight with anti-CD3 and anti-CD28 antibodies and IL-2, thentransduced with the lentivirus preparations at a multiplicity ofinfection of 5:1. The lentiviral vectors encoding the control 2^(nd)generation CAR was only used to transduce cells from one donor.

After two weeks (14 days) of culture, the cells were analyzed forcytotoxicity following co-culture with target antigen-expressing cellsusing the Acea Real-Time Cell Analyzer (RTCA), which measures theimpedance variations in the culture media of a 96-well microelectronicplate (E-plate), and shows the changes in cell number and morphology ina real-time plot. CD19-expressing HeLa target cells (HeLa-CD19) wereseeded into a 96-well E-plate and the impedance of each monolayer wasmonitored for 24 hours using the RTCA system. The engineered T cellswere added to the wells at an effector:target ratio of 10:1 and thewells were monitored for another 48 hours. The results were displayedand recorded as Cell Index (CI) value derived from the change inmeasured electrical impedance and were then ratio transformed bydividing the CI readouts of all wells at all time points over the CIvalue of individual wells at a same time (base-time) to obtain anormalized cell index value representing the percentage of the value atthe base-time (see Zhang et al. “Introduction to the Data Analysis ofthe Roche xCELLigence®System with RTCA Package.” Bioconductor. May, 3,2016,bioconductor.org/packages/devel/bioc/vignettes/RTCA/inst/doc/aboutRTCA.pdf.Accessed Sep. 9, 2016). In this assay, a decrease in the impedance of amonolayer reflects killing of the target cells by the transduced cells.

The results showed that decreased impedance was observed in cellsexpressing the 1^(st) generation CAR compared to non-transduced T cells,although the degree of decreased impedance for cells expressing the1^(st) generation CAR was less than cells expressing the 2^(nd)generation CAR. The decreased impedance in cells expressing the 1^(st)generation CAR continued generally for up to the first 8 hours of theassay, while only the 2^(nd) generation CAR-expressing cells continuedto decrease the impedance thereafter.

As shown in FIG. 1, in one donor, each of the cells co-expressing theTIP or corresponding wild-type transmembrane protein with the 1^(st)generation CAR exhibited a greater decrease in impedance, indicatinggreater cytotoxic activity, compared to cells only expressing the 1^(st)generation CAR. Further, the results showed that the cytotoxic activitywas greater in CAR-expressing cells that co-expressed the CD80-TIP orICOSL-TIP relative to CAR-expressing cells that co-expressed thecorresponding wild-type CD80 or ICOSL transmembrane proteins containinga non-affinity modified IgSF domain. The observed results of theseTIP-engineered cells showed that cytotoxic activity in cellsco-expressing the CD80-TIP or ICOSL-TIP with the CAR exhibit increasedactivity to modulate the cytotoxic immune response of antigen-specific Tcells, such as the CAR-expressing T cells.

In the other two donors, the cells expressing the CD80-TIP did notresult in a greater decreased impedance compared to cells expressing thecorresponding wild-type CD80 transmembrane protein. In one donor, therewere not enough cells to transduce with the wild-type transmembraneprotein construct, although in this donor the ICOS-L TIP gave the bestcytotoxicity compared to the other constructs tested. In the otherdonor, the cells expressing the ICOS-L-TIP did not result in a greaterdecreased impedance compared to cells expressing the correspondingwild-type ICOS-L transmembrane protein. In the tested cells, all cellsco-expressing either a CD80-TIP, ICOSL-TIP or corresponding wild typetransmembrane protein with the CAR exhibited greater cytotoxic activitythan cells only expressing the 1st generation CAR. The differences inthe results observed among donors may be related to the differences inthe T cells among the donors, differences in expression levels of thevarious engineered proteins on the surface of the cells, the particularconditions used in this exemplary assay for assessing killing in cells(e.g. assessing Day 14 transduced cells, assessing a singleeffector:target cell ratio) or other factors.

Example 10 Assessment of Binding and Activity of ICOSL IgSF DomainVariants

Additional ECD ICOSL variants were identified by the yeast selectionmethod substantially as described above and were used to produce ECD-Fcfusion proteins as described in Example 5. Binding studies wereperformed to assess specificity and affinity of ICOSL domain variantimmunomodulatory proteins for cognate binding partners substantially asdescribed in Example 6.

A. Binding and Functional Characterization

Binding was assessed to cells expressed full-length cognate bindingpartners CD28, ICOS and CTLA-4 substantially as described in Example 6.Bioactivity of the ECD ICOSL variants also was assessed in an anti-CD3coimmobilization assay or human Mixed Lymphocyte Reaction (MLR)substantially as described in Example 6, except that for thecoimmobilization assay, costimulatory activity was determined by cultureof human T cells with a mixture of 10 nM plate-bound anti-CD3 and 40 nMICOSL Fc variant proteins.

Table 15 depicts exemplary results for the additional ICOSL IgSF domainvariants for binding to cell-expressed counter structures andbioactivity in the anti-CD3 coimmobilization assay or MLR assay. Theexemplary amino acid substitutions depicted in Table 15 are designatedby amino acid position number corresponding to the respective referenceunmodified ICOSL ECD sequence set forth in SEQ ID NO:32. The amino acidposition is indicated in the middle, with the corresponding unmodified(e.g. wild-type) amino acid listed before the number and the identifiedvariant amino acid substitution listed after the number. Column 2 setsforth the SEQ ID NO identifier for the variant ECD for each variantECD-Fc fusion molecule.

The results in Table 15 depict binding activity as measured by the MeanFluorescence Intensity (MFI) value for binding of each variant Fc-fusionmolecule to cells engineered to express the cognate counter structureligand and the ratio of the MFI compared to the binding of thecorresponding unmodified ECD-Fc fusion molecule not containing the aminoacid substitution(s) to the same cell-expressed counter structureligand. The functional activity of the variant Fc-fusion molecules tomodulate the activity of T cells also is shown based on the calculatedlevels of IFN-gamma in culture supernatants (pg/ml) generated either i)with the indicated variant ECD-Fc fusion molecule coimmoblized withanti-CD3 or ii) with the indicated variant ECD-Fc fusion molecule in anMLR assay. The Table also depicts the ratio of IFN-gamma produced byeach variant ECD-Fc compared to the corresponding unmodified (parental)ECD-Fc in both functional assays.

The results show altered, including increased, binding affinity ofaffinity-modified ICOSL IgSF domain variants for at least one cognatecounter structure ligand and/or improved immunological activity.Specifically, similar to the initial hits identified in Example 6, theselections resulted in the identification of a number of additionalICOSL IgSF domain variants that were affinity-modified to exhibitincreased binding for at least one, and in some cases more than one,cognate counter structure ligand. In addition, the results showed thataffinity modification of the variant molecules also exhibited improvedactivities to both increase and/or decrease immunological activitydepending on the format of the molecule as described in Example 6.

TABLE 15 ICOSL variants: binding data and costimulatory bioactivitydata. Anti-CD3 IFN-gamma Coimmobil- MLR ICOS tfxn CD28 tfxn CTLA-4ization IFN-gamma SEQ MFI MFI tfxn MFI Assay pg/ml pg/ml ID NO (parental(parental (parental (parental (parental ICOSL mutation(s) (ECD) ratio)ratio) ratio) ratio) ratio) N52H, F78L, Q100R, C198R 373 9568 1966 1454130 5927 (0.12) (0.24) (0.12) (0.31) (1.84) N52H, N57Y, Q100R, 364 9418136665 115352 944 821 V110D, C198R, S212G (1.16) (16.55) (9.59) (2.21)(0.25) N52H, N57Y, R75Q, Q100P, 374 5558 7465 4689 122 1136 V110D (0.07)(0.90) (0.39) (0.28) (0.35) N52H, N57Y, Q100R, C198R 365 9148 13492383241 1060 375 (1.13) (16.33) (6.92) (2.48) (0.12) N52H, N57Y, L74Q,V110D, 375 9448 128342 123510 1137 889 S192G (1.17) (15.54) (10.26)(2.66) (0.28) N52H, Q100R 285 9478 151977 133929 972 794 (1.17) (18.40)(11.13) (2.28) (0.25) N52H, S121G, C198R 376 9128 124732 182607 827 1257(1.13) (15.10) (15.18) (1.94) (0.39) A20V, N52H, N57Y, Q100R, 287 582876973 73640 447 2283 S109G (0.72) (9.32) (6.12) (1.05) (0.71) N52H,N57Y, Q100P, C198R 461 9548 130676 81966 1125 643 (1.18) (15.82) (6.81)(2.64) (0.20) N52H, N57Y, R61S, Q100R, 289 1018 9129 5790 109 5094V110D, L173S (0.13) (1.11) (0.48) (0.25) (1.58) N52H, N57Y, Q100R, 2909978 137372 70764 1316 473 V122A (1.23) (16.63) (5.88) (3.08) (0.15)N52H, N57Y, Q100R, F172S 291 1028 135821 73320 1561 486 (1.27) (16.44)(6.09) (3.66) (0.15) N52H, N57Y, Q100R 283 9858 140612 75106 1648 778(1.22) (17.02) (6.24) (3.86) (0.24) N52S, F120S, N227K 377 9438 6779682370 1157 1626 (1.17) (8.21) (6.85) (2.71) (0.50) N52S, N194D 366 979859431 74502 1671 1690 (1.21) (7.19) (6.19) (3.91) (0.52) N52S, V97A 2943138 1733 1541 84 3858 (0.04) (0.21) (0.13) (0.20) (1.20) N52S, F120S293 9068 67233 97880 1178 2814 (1.12) (8.14) (8.13) (2.76) (0.87) N52S,G72R 295 9288 51638 62339 1161 2947 (1.15) (6.25) (5.18) (2.72) (0.91)N52S, A71T, A117T, T190A, 378 8918 44044 56646 1076 4031 C198R (1.10)(5.33) (4.71) (2.52) (1.25) N52S, E220G 297 3878 2047 1796 122 1927(0.05) (0.25) (0.15) (0.29) (0.60) Y47H, N52S, V107A, F120S 298 32682562 2104 334 4390 (0.04) (0.31) (0.17) (0.78) (1.36) WT ICOSL 32 80888260 12033 427 3226 (1.00) (1.00) (1.00) (1.00) (1.00) T43A, N52H, N57Y,L74Q, 379 2821 2180 2051 184 D89G, V110D, F172S (0.02) (0.49) (0.12)(0.75) N52H, N57Y, Q100R, V107I, 381 174586 122383 76202 985 1037 V110D,S132F, I154F, (0.97) (27.24) (4.31) (4.01) (0.36) C198R, R221G E16V,N52H, N57Y, Q100R, 300 190765 129070 68488 4288 1225 V110D, H115R,Y152C, (1.05) (28.73) (3.87) (17.46) (0.43) K156M, C198R Q37R, N52H,N57Y, Q100R, 301 148638 91104 13498 62 7643 V110N, S142F, C198R, (0.82)(20.28) (0.76) (0.25) (2.68) D217V, R221G N52H, N57Y, Q100R, 302 179194123312 84136 762 1342 V110D, C198R (0.99) (27.45) (4.76) (3.10) (0.47)N52H, N57Y, Q100R, 303 5236 4160 3305 49 2039 V110D, V116A, L161M,(0.03) (0.93) (0.19) (0.20) (0.72) F172S, S192G, C198R F27S, N52H, N57Y,V110N 304 20154 8613 3903 83 7522 (0.11) (1.92) (0.22) (0.34) (2.64)F27S, N52H, N57Y, V110N 304 5236 4160 2957 40 — (0.03) (0.93) (0.17)(0.16) N52S, H94E, L96I, S109N, 305 198604 100361 102892 1253 5645L166Q, (1.10) (22.34) (5.82) (5.10) (1.98) S18R, N52S, F93L, I143V, 306154561 7625 4254 203 5239 R221G (0.85) (1.70) (0.24) (0.83) (1.84) A20T,N52D, Y146C, Q164L 307 149661 9073 6901 287 4829 (0.83) (2.02) (0.39)(1.17) (1.69) V11E, N30D, N52H, N57Y, 308 180016 120230 62809 2218 7283H94E, L96I, L98F, N194D, (1.00) (26.76) (3.55) (9.03) (2.56) V210A,1218T N52S, H94E, L96I, V122M 309 198717 88901 94231 590 618 (1.10)(19.79) (5.33) (2.40) (0.22) N52H, N57Y, H94E, L96I, 310 87711 4203531798 67 2500 F120I, S126T, W153R, (0.48) (9.36) (1.80) (0.27) (0.88)I218N M10V, S18R, N30D, N52S, 311 180665 64929 48362 1193 13647 S126R,T139S, L203F (1.00) (14.45) (2.73) (4.86) (4.79) S25G, N30D, N52S, 312178834 66127 46631 1246 2202 F120S, N227K (0.99) (14.72) (2.64) (5.07)(0.77) N30D, N52S, L67P, Q100K, 313 18630 1986 1940 54 2752 D217G,R221K, T225S (0.10) (0.44) (0.11) (0.22) (0.97) WT ICOSL 32 180900 449317685 246 2850 (1.00) (1.00) (1.00) (1.00) (1.00) N52H, N57Y, Q100R, 3142831 2881 2464 59 — V110D, A117T, T190S, (0.04) (0.57) (0.23) (0.08)C198R N52H, N57Y, Q100R, 315 58478 74031 56850 712 1093 V110D, F172S,C198R (0.79) (14.75) (5.33) (0.96) (0.23) S25G, F27C, N52H, N57Y, 31622514 21320 20450 353 5765 Q100R, V110D, E135K, (0.30) (4.25) (1.92)(0.48) (1.21) L173S, C198R N52H, N57Y, V110A, 317 84236 81842 1215194593 1137 C198R, R221I (1.14) (16.31) (11.39) (6.18) (0.24) M10I, S13G,N52H, N57Y, 318 6362 6001 4834 141 4326 D77G, V110A, H129P, (0.09)(1.20) (0.45) (0.19) (0.91) I143V, F172S, V193M, C198R N52H, N57Y, R61C,Y62F, 319 4355 4316 3430 110 6854 Q100R, V110N, F120S, (0.06) (0.86)(0.32) (0.15) (1.44) C198R N52H, N57Y, Q100R, 367 96736 77881 1480128765 630 L102R, V110D, H115R, (1.31) (15.52) (13.88) (11.79) (0.13)C198R N52H, N57Y, Q100R, 321 67578 64953 95731 1672 1490 V110D, N144D,F172S, (0.91) (12.94) (8.98) (2.52) (0.31) C198R N52S, H94E, L98F, 32280690 78750 148160 3564 1497 Q100R, (1.09) (15.69) (13.89) (4.80) (0.32)N52S, E90A 323 108908 31086 108866 4564 3927 (1.47) (6.19) (10.21)(6.14) (0.83) N30D, K42E, N52S 324 85726 4293 10755 5211 5656 (1.16)(0.86) (1.01) (7.01) (1.19) N52S, F120S, I143V, 325 90862 28443 1052294803 4357 I224V (1.23) (5.67) (9.87) (6.46) (0.92) WT ICOSL 32 739645018 10665 743 4748 (1.00) (1.00) (1.00) (1.00) (1.00)

B. Cytokine Production in Anti-CD3 Costimulation Assays

Exemplary variant ECD ICOSL Fc-fusion molecules described above werefurther assessed for stimulation of cytokines IL-17 in the anti-CD3costimulatory (coimmobilization) bioactivity assay described above. Amixture of 10 nM plate-bound anti-CD3 and 40nM ICOSL Fc variant proteinswere cultured with human T cells. Supernatants were collected and IL-17levels were determined by ELISA. The amount of IL-17 in culturesupernatants (pg/ml) generated with the indicated variant ECD-Fc fusionmolecule and corresponding unmodified (parental) ECD-Fc coimmobilizedwith anti-CD3 was measured. For comparison, also shown in this Table arethe results for production of IFN-gamma in the same assay as depicted inTable 15 for the exemplary variants.

Results are shown in Table 16, which depict the pg/mL of IL-17 measuredin the supernatant as well as the ratio (fold increase) of IL-17produced by each variant ECD-Fc compared to the corresponding unmodified(wild-type) ECD-Fc. Similar results are shown for IFN-gamma. Also shownis the % of total IL-17 or IFN-gamma cytokine produced by cells. Theresults showed that affinity modification of the variant moleculesexhibited altered functional T cell activity to increase IL-17 inaddition to IFN-gamma in the costimulation assay.

TABLE 16 Costimulatory Bioreactivity Data for ICOSL IgSF Domain Variants% of Total Cytokine % SEQ IL-17A IFN-g Total Produced Total ID NO IL-17AFold IFN-g Fold Fold % % IL-17 + ICOSL mutation(s) (ECD) [pg/mL] ↑WT[pg/mL] ↑WT ↑WT IL-17 IFN-g IFN-g N52H, N57Y, 365 617 7.93 1060 2.4810.42 5.51 0.77 6.28 Q100R, C198R N52H, N57Y, 290 647 8.33 1316 3.0811.41 5.79 0.96 6.75 Q100R, V122A N52H, N57Y, 291 549 7.06 1561 3.6610.72 4.91 1.14 6.05 Q100R, F172S N52Y, N57Y, 112 90 1.05 1999 2.69 3.740.81 2.91 3.72 F138L, L203P V11E, N30D, 308 319 3.16 2218 9.03 12.192.85 3.23 6.08 N52H, N57Y, H94E, L96I, L98F, N194D, V210A, I218T N52H,N57Y, 367 510 5.90 8765 11.79 17.70 4.56 12.78 17.33 Q100R, L102R,V110D, H115R, C198R N52H, N57Y, 283 473 6.08 1648 3.86 9.94 4.23 1.205.43 Q100R N52H, Q100R 285 358 4.60 972 7.01 11.62 3.20 0.71 3.91 N52H,N57Y, 364 124 1.60 944 2.21 3.81 1.11 0.69 1.80 Q100R, V110D, C198R,S212G N52H, N57Y, 113 127 1.47 4922 6.62 8.09 1.14 7.17 8.31 Q100P E16V,N52H, 300 22 7.11 130 17.46 24.57 6.41 6.25 12.66 N57Y, Q100R, V110D,H115R, Y152C, K156M, C198R N30D, K42E, 324 349 4.04 5211 7.01 11.05 3.127.60 10.71 N52S N52S, F120S, 325 292 3.39 4803 6.46 9.85 2.61 7.00 9.62I143V, I224V N52S, E90A 323 306 3.54 4564 6.14 9.68 2.73 6.65 9.39 N52H,N57Y, 317 290 3.35 4593 6.18 9.53 2.59 6.69 9.28 V110A, C198R, R221IN52S, N194D 366 428 5.50 1671 3.90 9.4 1.52 5.19 5.40 N52H, I143T 135 84— 1727 — 3.30 0.75 2.52 3.27 N52D 111 126 — 1447 — 3.41 1.13 2.11 3.23

Example 11 Generation of Additional Engineered T cell Expressing aTransmembrane Immunomodulatory Protein and Assessment of Proliferation

This Example describes the generation of additional engineered T cellsin which a transmembrane immunomodulatory protein (TIP) containing anextracellular domain (ECD) containing ICOSL affinity-modified IgSFdomain was co-expressed with a chimeric antigen receptor (CAR).Specifically, the TIP was generated to include the ECD of exemplaryvariant ICOSL containing amino acid mutations N52D, N52H/N57Y/Q100P,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R or N52H/N57Y/Q100Rwith reference to positions in the ICOSL extracellular domain set forthin SEQ ID NO:32. The TIP also contained a transmembrane domain and acytoplasmic domain of the corresponding wild-type ICOSL transmembraneprotein sequence corresponding to residues 257-302 of SEQ ID NO:5. Thesequence of the TIP with and without its signal peptide are as follows:N52D (SEQ ID NO: 496 and 497); N52H/N57Y/Q100P (SEQ ID NO: 498 and 499);E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R (SEQ ID NO: 500 and501) and N52H/N57Y/Q100R (SEQ ID NO: 502 and 503). For comparison, thefull-length transmembrane wild-type ICOSL (amino acid residues 19-302 ofSEQ ID NO:5) also was expressed in cells. The sequence of the wildtypeTIP with and without its signal peptide is set forth in SEQ ID NO:494and 495. The nucleic acid encoding the TIP also included a sequenceencoding a green fluorescent protein (GFP) separated from the TIP by aself-cleaving T2A sequence.

The TIP containing the affinity-modified domain or the wild-typetransmembrane protein containing a corresponding non-affinity modifiedIgSF domain were co-expressed in T cells with a chimeric antigenreceptor (CAR). The nucleotide sequence encoding the CAR encodes, inorder: a CD8 signal sequence (SEQ ID NO:481), an anti-CD19 scFv (SEQ IDNO:482), a hinge/transmembrane region derived from CD8 (SEQ ID NO:483),a costimulatory signaling domain derived from 4-1BB (SEQ ID NO:484), anda CD3zeta signaling domain (SEQ ID NO:247). The resulting anti-CD19 CARhas the sequence of amino acids set forth in SEQ ID NO:490. The nucleicacid encoding the CAR also included a sequence encoding a bluefluorescent protein (BFP; SEQ ID NO:489) separated from the CAR by aself-cleaving T2A sequence (set forth in SEQ ID NO:488).

Viral vector constructs were separately generated into which was clonedeither the nucleic acid molecule encoding the CAR alone or a nucleicacid molecule encoding one of the exemplary TIPs or wild-type ICOSL. Theviral vector encoding the CAR and the viral vector encoding the TIP orwild-type ICOSL were co-transduced into T cells. For transduction,primary T cells were activated with anti-CD3 and anti-CD28 beads (Dynal)at 1:1 bead:cell ratio and incubated in the presence of 100 IU/mL ofIL-2 at 37° C. for 2 days. T cells were then harvested and transducedwith 400 μL of CAR viral supernatant and 400 μL of TIP viral supernatantin the presence of 8 μg/mL of polybrene. The cells were spinoculated at1000 g for 30 minutes at 30° C. The cells were then transferred andincubated overnight at 37° C. After the incubation, cells were collectedand viral supernatant was removed. The cells were resuspended withcomplete media and 50 IU/mL of IL-2. Cells were expanded, replenishedwith IL-2 and media every two days for a total of 6 days. Beads wereremoved from the cells using a magnet and counted before being assessedin a proliferation assay. An exemplary expression profile of a TIP andCAR in an exemplary transduced T cells is shown in FIG. 2A.

To assess proliferation of CAR T cells and CAR-TIP T cells in responseto antigen, cells were labeled with cell trace far red dye.CD19-expressing Nalm6 target cells were titrated starting from 1.5:1target:T cell ratio and by 1:2 dilutions with 8-point dilution. LabeledCAR T cells or CAR-TIP T cells were added to Nalm6 cells and the culturewas incubated for 4 days before cells were analyzed by flow cytometry.The supernatant was collected and further assessed in a cytokine releaseassay.

As shown in FIG. 2B, CAR+ primary T cells proliferate in a dosedependent manner to CD19+ NALM6 cells. Compared to a CAR only T cells, Tcells coexpressing the CAR and either wild-type ICOSL or one of theexemplary ICOSL TIP exhibited enhanced proliferation compared to CARonly expressing T cells. Co-expression of a CAR and a TIP containingeither the N52H/N57Y/Q100P,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R or N52H/N57Y/Q100Rvariant ICOSLECD exhibited greater proliferation than T cellsco-expressing the CAR and wildtype ICOSL, indicating that TIPs expressedon primary T cells provide an improved costimulatory signal to enhance Tcell proliferation.

Example 12 Purification and Assessment of Purified of ICOSL IgSF DomainVariants

A purification strategy was employed for exemplary candidate hitsdescribed in Example 6 and 10. Human cells derived from the 293 cellline (Expi293) were transiently transfected with expression constructand the ECD ICOSL Fc fusion molecule was expressed in the cells. The Fcfusion proteins were then purified from supernatants with Protein A byaffinity chromatograpy (MabSelect SuRe). This initial purification stepwas then followed by a preparative size exclusion chromatography (SEC)step to further purify the proteins (Superdex200 16×60). Samples fromboth purification steps were retained and compared by analytic SEC. Theconcentration of the protein was determined after Protein Apurification. The resulting purified proteins also were analyzed byanalytic SEC on a high performance liquid chromatography (HPLC) toassess purity.

The percent main peak in the purified samples was determined andcompared to protein purified in the initial Protein A step (% Main PeakProt A pool) versus protein purified with Protein A followed bypreparative SEC (% Main Peak SEC pool T=DO). As shown in Table 17, theadditional SEC step substantially increased protein purity of purifiedproteins. To further assess stability of the proteins, proteins purifiedby preparative SEC were left at room temperature for 24 hours and then %Main Peak by HPLC (% Main Peak SEC pool T=D24) was assessed and comparedto D0 sample. The change in % Main Peak at DO versus D24 was determined(▴% Main Peak SEC pool). As shown in Table 17, most of the testedexemplary variant ECD ICOSL Fc fusion molecules exhibited little changein % Main Peak at this time, indicating minimal aggregation of theprotein variants had occurred.

TABLE 17 Purification of ICOSL Protein Variants % % % Main Main ▴ %Expi293 Main Peak Peak Main SEQ Prod. Peak SEC SEC Peak ID NO Prot AProt A pool pool SEC ICOSL mutation(s) (ECD) mg/L pool T = D 0 T = D 24pool N52S, N194D 366 120 87.9 93.5 92 1.5 N52H, N57Y, Q100R, F172S 291217 86.9 97.4 95.6 1.8 N52S, E90A 323 128 86.5 89.5 88.3 1.2 N52H, Q100R285 176 85.9 97.5 96.1 1.4 N52H, N57Y, Q100R 283 186 85.1 97.6 95.7 1.9N52S, F120S, I143V, I224V 325 87 83.2 88.9 88.3 0.6 N52H, N57Y, Q100R,C198R 365 204 82.9 95.8 92.3 3.5 N52H, N57Y, Q100P 113 63 80.5 94.5 88.56 N30D, K42E, N52S 324 81 80 95.4 91.3 4.1 N52H, N57Y, Q100R, L102R, 367141 78.9 96 92.9 3.1 V110D, H115R, C198R N52H, N57Y, Q100R, V122A 290260 77.6 96.4 95.2 1.2 N52Y/N57Y/F138L/L203P 112 40 75.6 96.8 94.8 2E16V, N52H, N57Y, Q100R, 300 60 73.8 97.1 95.8 1.3 V110D, H115R, Y152C,K156M, C198R N52H, N57Y, V110A, C198R, 317 95 65.4 90.9 86 4.9 R221IN52H, N57Y, Q100R, V110D, 364 73 50.6 87.9 78.6 9.3 C198R, S212G V11E,N30D, N52H, N57Y, 308 58 — — — — H94E, L96I, L98F, N194D, V210A, I218TN52H, I143T 135 134 93.2 96 92.7 3.3 N52D 111 136 90.4 95.5 93.3 2.2

Example 13 Assessment of Costimulatory Bioactivity of Purified ICOSLIgSF Domain Variant Hits

Exemplary ECD ICOSL Fc fusion molecules purified as described in Example12 were assessed for bioactivity by MLR substantially as described inExample 6. A mixture of 10 nM or 40 nM ICOSL Fc variant proteins wasbound overnight to 96-well plates in the presence of 10 nM anti-CD3. Theplates were washed and 100,000 CFSE labelled pan T cells were added for96 hours. Supernatants were collected, and IFN-gamma and IL-17 levelswere measured by ELISA.

Results for the cytokine secretion induced by anti-CD3 costimulationwith exemplary tested variants (10 nM and 40 nM ICOSL Fc) are shown inFIG. 3A and 3B, which indicates exemplary IgSF domain amino acidsubstitutions (replacements) in the ECD of ICOSL. The bar graphs in FIG.3A and 3B depict the amount of secreted IFN-gamma and IL-17,respectively, by ELISA in the supernatants (pg/mL). The level ofcytokine release induced by anti-CD3 costimulation with the testedvariants compared to the level induced by anti-CD3 costimulation with WTICOSL is indicated by the horizontal line. The results showed thataffinity modification of the variant molecules exhibited activity tomodulate functional T cell activity, including to substantially increaseIFN-gamma and IL-17 secretion in the costimulation assay. Increasedimmunological activity was observed with some variants.

Example 14 Assessment of Proliferation of Purified ICOSL IgSF DomainVariant Hits

Exemplary variant ECD ICOSL Fc fusion molecules purified as described inExample 12 were assessed for ability to costimulate anti-CD3-inducedproliferation of T cells. Primary T cells were labeled withcarboxyfluorescein succinmidyl ester (CFSE). A mixture of 10 nM or 40 nMvariant ECD ICOSL Fc or wild-type ECD ICOSL proteins were boundovernight to 96-well plates in the presence of 10 nM anti-CD3, and thenlabeled T cells were added and incubated for 3 days. As a control,proliferation also was assessed in the presence of bound anti-CD3 andIgG or IgG alone. Cells were stained for CD4 or CD8 surface markers andproliferation of total T cells, CD4+ T cells or CD8+ T cells wasdetermined by assessing CFSE dilution by flow cytometry.

The results are set forth in FIG. 4A and FIG. 4B for exemplary variantstested at 40 nM and 10 nM ICOSL, respectively. As shown in FIG. 4A,nearly all tested variant ECD ICOSL Fc fusion molecules inducedproliferation greater than WT control. As shown in FIG. 4B, differencesin proliferation were more apparent at 10 nM with certain variantsproviding maximal proliferation even at this lower concentration.

Example 15 Assessment of Binding and Activity of Purified ICOSL IgSFDomain Variant Hits

Exemplary variant ECD ICOSL Fc fusion molecules purified as described inExample 12 were assessed for binding and functional activities usingmethods substantially as described in Example 6 or Example 10.

A. Flow Cytometric Binding Assays

Human cells derived from the 293 cell line (Expi293) were transfectedwith CD28, CTLA-4, ICOS or mock transfected. Cells were then incubatedwith ECD ICOSL Fc fusion molecules or wild-type ECD ICOSL-Fc that weretitrated from 100,000 pM to 46 pM, and binding was observed using aPE-conjugated anti-human Fc as described in Example 6. Binding wasassessed by flow cytometry and mean fluorescence intensity (MFI) andpercent (%) of cells positive for signal was determined using Cell QuestPro software (Becton Dickinson, USA). The concentration of ICOSL-Fc thatgave a half-maximal MFI response (MFI EC50) or % positive cells (% (+)EC50) was determined.

Table 18 sets forth the results. The ICOSL amino acid substitutionsdepicted in Table 18 are designated by amino acid position numbercorresponding to the respective reference unmodified ICOSL ECD sequenceset forth in SEQ ID NO:32. For some values (e.g. WT binding to CD28) itwas not possible to obtain an EC50, therefore 1000000 pM was arbitrarilypiced for data formatting purposes. Similar to results obtained fromprevious binding assays as described in Example 10 above, alteredbinding affinity of variant ICOSL ECD-Fc fusion molecule for at leastone cognate counter structure ligand was observed.

TABLE 18 Flow Cytometric EC50s for ICOSL variants CD28 CD28 CTLA-4CTLA-4 ICOS ICOS SEQ MFI % (+) MFI % (+) MFI % (+) ID NO EC50 EC50 EC50EC50 EC50 EC50 ICOSL mutation(s) (ECD) [pM] [pM] [pM] [pM] [pM] [pM] WTICOSL 32 1000000 1000000 1000000 1000000 10543 762 N52H, I143T 135 19147567 20259 1891 2666 286 N52H, N57Y, Q100R, 365 950 159 73548 422 1032179 C198R N52H, N57Y, Q100R, V122A 290 29701 152 1008 293 302 64 N52H,N57Y, Q100R, F172S 291 1006 231 1332 396 779 130 N52Y/N57Y/F138L/L203P112 7844 386 7457 994 3104 408 V11E, N30D, N52H, N57Y, 308 5961 595 69091026 5514 852 H94E, L96I, L98F, N194D, V210A, I218T N52H, N57Y, Q100R,L102R, 367 1034 307 23328 579 3172 347 V110D, H115R, C198R N52H, N57Y,Q100R 283 1665 238 11002 533 383 131 N52H, Q100R 285 1305 274 8593 1997702 167 N52H, N57Y, Q100R, V110D, 364 4987 594 30382 922 50219 814C198R, S212G N52H, N57Y, Q100P 113 21137 402 22651 758 4090 320 E16V,N52H, N57Y, Q100R, 300 2508 387 5399 806 2381 421 V110D, H115R, Y152C,K156M, C198R N30D, K42E, N52S 324 — 3683800 8593 1997 3251 558 N52S,F120S, I143V, I224V 325 902400 9060 28126 2948 4366 245 N52S, E90A 3231339700 31302 31419 5828 5225 473 N52H, N57Y, V110A, C198R, 317 1809 4267201 841 1293 433 R221I N52S, N194D 366 944669 11876 1254880 5170 473206 N52D 111 288617 17793 396841 3891 2642 137

B. ForteBio Binding Assay

Protein-protein interactions between the receptors and ICOSL domainvariant immunomodulatory proteins were further assessed using Fortebiobinding assays. ICOS, CD28, and CTLA-4 receptors were loadedindividually onto anti-human capture sensors (ForteBio Octet AHC) andwildtype unmodified ICOSL ECD-Fc fusion molecule, wildtype PD-L2 ED-Fcfusion molecule or variant ICOSL Fc-fusion molecules were bound to thereceptors in 4 point titrations. Each titration was globally fit tocalculate the associate (k_(on)) and dissociation (K_(dis)) of eachprotein. Loading response of anti-human capture sensors of each receptorbeing tested with the variant ICOSL ECD-Fc fusion molecule wasdetermined. The dissociation constant (KD) was calculated and comparedto wildtype to determine a fold improvement value (fold imp.).

Binding results to ICOS are set forth in Table 19, to CD28 are set forthin Table 20 and to CTLA-4 are set forth in Table 21. The exemplary aminoacid substitutions depicted in Table 19-21 are designated by amino acidposition number corresponding to the respective reference unmodifiedICOSL ECD sequence set forth in SEQ ID NO:32.

TABLE 19 ICOS ForteBio Binding Assay SEQ ID NO K_(on) K_(dis) Fold ICOSLmutation(s) (ECD) Response KD (M) (1/Ms) (1/s) Full R² Imp. WT ICOSL 320.73 8.83E−10 1.78E+05 1.58E−04 0.9908 — N52H, I143T 135 0.87 3.32E−103.13E+05 1.04E−04 0.9683 2.7 N52H, N57Y, Q100R, 365 0.74 4.92E−103.85E+05 1.89E−04 0.9882 1.8 C198R N52H, N57Y, Q100R, 290 0.67 4.72E−103.77E+05 1.78E−04 0.9775 1.9 V122A N52H, N57Y, Q100R, 291 0.68 4.20E−104.34E+05 1.82E−04 0.9545 2.1 F172S N52Y/N57Y/F138L/L203P 112 0.647.69E−10 2.22E+05 1.71E−04 0.9782 1.1 V11E, N30D, N52H, 308 0.673.62E−10 3.55E+05 1.29E−04 0.9687 2.4 N57Y, H94E, L96I, L98F, N194D,V210A, I218T N52H, N57Y, Q100R, 367 0.76 4.77E−10 3.29E+05 1.57E−040.9616 1.9 L102R, V110D, H115R, C198R N52H, N57Y, Q100R 283 0.743.69E−10 2.87E+05 1.06E−04 0.9817 2.4 N52H, Q100R 285 0.79 3.73E−104.45E+05 1.66E−04 0.968 2.4 N52H, N57Y, Q100R, 364 0.60 1.29E−091.66E+05 2.15E−04 0.9846 0.7 V110D, C198R, S212G N52H, N57Y, Q100P 1130.73 3.82E−10 3.71E+05 1.42E−04 0.9729 2.3 E16V, N52H, N57Y, 300 0.755.43E−10 2.65E+05 1.44E−04 0.9848 1.6 Q100R, V110D, H115R, Y152C, K156M,C198R N30D, K42E, N52S 324 0.80 3.71E−10 4.48E+05 1.66E−04 0.9651 2.4N52S, F120S, I143V, 325 0.80 3.11E−10 5.03E+05 1.56E−04 0.9673 2.8 I224VN52S, E90A 323 0.88 3.40E−10 4.85E+05 1.65E−04 0.9792 2.6 N52H, N57Y,V110A, 317 0.68 4.77E−10 3.15E+05 1.50E−04 0.976 1.9 C198R, R221I N52S,N194D 366 0.88 3.37E−10 3.38E+05 1.14E−04 0.9723 2.6 N52D 111 0.873.38E−10 3.91E+05 1.32E−04 0.9792 2.6 Wildtype PD-L2 ED-Fc — 0.03

TABLE 20 CD28 ForteBio Binding Assay SEQ ID NO K_(on) K_(dis) Fold ICOSLmutation(s) (ECD) Response KD (M) (1/Ms) (1/s) Full R² Imp. WT ICOSL 320.33 1.39E−08 6.69E+04 9.29E−04 0.9715 — N52H, I143T 135 0.95 5.25E−104.27E+05 2.24E−04 0.9877 26.5 N52H, N57Y, Q100R, 365 1.14 4.47E−104.12E+05 1.84E−04 0.9877 31.0 C198R N52H, N57Y, Q100R, 290 1.04 3.90E−104.07E+05 1.59E−04 0.9878 35.6 V122A N52H, N57Y, Q100R, 291 1.06 2.93E−104.26E+05 1.25E−04 0.9836 47.3 F172S N52Y/N57Y/F138L/L203P 112 0.867.83E−10 1.79E+05 1.40E−04 0.993 17.7 V11E, N30D, N52H, 308 0.925.53E−10 2.54E+05 1.40E−04 0.9906 25.1 N57Y, H94E, L96I, L98F, N194D,V210A, I218T N52H, N57Y, Q100R, 367 1.10 3.66E−10 3.41E+05 1.25E−040.986 37.9 L102R, V110D, H115R, C198R N52H, N57Y, Q100R 283 1.043.68E−10 3.72E+05 1.37E−04 0.983 37.7 N52H, Q100R 285 1.09 4.01E−105.0SE+05 2.02E−04 0.9938 34.7 N52H, N57Y, Q100R, 364 0.94 8.96E−101.78E+05 1.60E−04 0.9961 15.5 V110D, C198R, S212G N52H, N57Y, Q100P 1130.99 4.36E−10 3.29E+05 1.43E−04 0.9835 31.8 E16V, N52H, N57Y, 300 1.065.03E−10 3.06E+05 1.54E−04 0.9872 27.6 Q100R, V110D, H115R, Y152C,K156M, C198R N30D, K42E, N52S 324 0.54 1.95E−09 2.74E+05 5.33E−04 0.97727.1 N52S, F120S, I143V, 325 0.84 9.10E−10 4.51E+05 4.10E−04 0.9742 15.3I224V N52S, E90A 323 0.94 9.69E−10 4.74E+05 4.59E−04 0.978 14.3 N52H,N57Y, V110A, 317 0.94 5.63E−10 2.63E+05 1.48E−04 0.9781 24.7 C198R,R221I N52S, N194D 366 0.82 1.04E−09 3.53E+05 3.68E−04 0.9887 13.3 N52D111 0.86 1.16E−09 3.36E+05 3.90E−04 0.989 11.9 wildtype PD-L2 ED-Fc —−0.04

TABLE 21 CTLA-4 ForteBio Binding Assay SEQ ID NO K_(on) K_(dis) FoldICOSL mutation(s) (ECD) Response KD (M) (1/Ms) (1/s) Full R² Imp. WTICOSL 32 0.21 7.71E−08 1.92E+04 1.48E−03 0.8919 — N52H, I143T 135 0.966.78E−10 7.26E+05 4.92E−04 0.9641 113.8 N52H, N57Y, Q100R, 365 1.576.45E−10 4.79E+05 3.09E−04 0.9875 119.6 C198R N52H, N57Y, Q100R, 2901.43 5.76E−10 4.73E+05 2.72E−04 0.9926 133.9 V122A N52H, N57Y, Q100R,291 1.47 5.36E−10 5.13E+05 2.75E−04 0.9924 144.0 F172SN52Y/N57Y/F138L/L203P 112 1.33 8.33E−10 3.45E+05 2.87E−04 0.9943 92.6V11E, N30D, N52H, 308 1.50 6.48E−10 3.12E+05 2.02E−04 0.9943 119.0 N57Y,H94E, L96I, L98F, N194D, V210A, I218T N52H, N57Y, Q100R, 367 1.608.64E−10 4.79E+05 4.14E−04 0.9825 89.2 L102R, V110D, H115R, C198R N52H,N57Y, Q100R 283 1.65 7.19E−10 4.28E+05 3.08E−04 0.9895 107.2 N52H, Q100R285 1.17 5.92E−10 8.37E+05 4.96E−04 0.9629 130.3 N52H, N57Y, Q100R, 3641.32 1.47E−09 2.34E+05 3.44E−04 0.9937 52.6 V110D, C198R, S212G N52H,N57Y, Q100P 113 1.51 6.47E−10 3.61E+05 2.33E−04 0.9911 119.2 E16V, N52H,N57Y, 300 1.58 1.06E−09 4.24E+05 4.49E−04 0.9779 72.8 Q100R, V110D,H115R, Y152C, K156M, C198R N30D, K42E, N52S 324 0.42 2.81E−09 2.42E+056.81E−04 0.9676 27.4 N52S, F120S, I143V, 325 0.58 1.20E−09 3.10E+053.72E−04 0.9283 64.3 I224V N52S, E90A 323 0.64 1.12E−09 3.28E+053.68E−04 0.9184 68.7 N52H, N57Y, V110A, 317 1.44 1.07E−09 4.0SE+054.32E−04 0.9811 72.3 C198R, R221I N52S, N194D 366 0.59 2.52E−09 2.66E+056.69E−04 0.9643 30.6 N52D 111 0.62 1.52E−09 4.16E+05 6.32E−04 0.923450.7 wildtype PD-L2 ED-Fc — 0.00

C. Coimmobilization Assay

Costimulatory bioactivity of ICOSL fusion variants was determined inanti-CD3 coimmobilization assays substantially as described in Example6. Approximately 0.37 nM, 1.3 nM or 10 nM mouse anti-human CD3 (OKT3,Biolegends, USA) was diluted in PBS with 10 nM or 40 nM variant ICOSLECD Fc vor wild-type ICOSL ECD-Fc. This mixture was added to tissueculture treated flat bottom 96 well plates overnight to facilitateadherence of the stimulatory proteins to the wells of the plate. Thenext day, unbound protein was washed off the plates and 100,000 purifiedhuman pan T cells were added to each well. Cells were cultured 3 daysbefore harvesting culture supernatants and measuring human IFN-gammalevels with an ELISA kit.

Table 22 sets fort the amount of IFN-gamma (pg/mL) produced by cellsunder the various conditions in the anti-CD3 coimmobilization assay. Inthe Table, the amino acid substitutions of exemplary variant ECDICOSL-Fc fusions are designated by amino acid position numbercorresponding to the unmodified ICOSL ECD sequence set forth in SEQ IDNO: 32 and the corresponding SEQ ID NO identifier for the variant ECDfor each variant ICOSL ECD-Fc fusion molecule is set forth. The ratio ofIFN-gamma produced in the presence of each variant ICOSL ECD-Fc in thefunctional assay compared to in the presence of the correspondingunmodified (wildtype) ECD-Fc is shown (Fold TWT). As shown,costimulatory signaling of the variant ICOSL-ECD-Fc molecules wassubstantially greater compared to wildtype ICOSL.

TABLE 22 Assessment of IFN-gamma Responses in Co-stimulation AssayIFN-gamma [pg/mL] SEQ 40 nM Ligand 10 nM Ligand 40 nM Ligand ID NO 10 mMFold 10 mM Fold 10 mM 1.1 nM 0.37 nM ICOSL mutation(s) (ECD) OKT3 ↑WTOKT3 ↑WT OKT3 OKT3 OKT3 E16V, N52H, N57Y, 300 14372 17.3 4903 29.98379.2 7422.8 2893.7 Q100R, V110D, H115R, Y152C, K156M, C198R N52H,N57Y, 290 10640 12.8 6456 39.4 5636.2 4724.2 2246.3 Q100R, V122A N52H,N57Y, 291 10379 12.5 3741 22.8 3979.7 4067.7 1415.5 Q100R, F172S N52H,N57Y, 365 9590 11.5 4048 24.7 4215.8 2787.1 1072.4 Q100R, C198R N52H,N57Y, Q100R 283 9568 11.5 3270 19.9 4412.3 3862.0 1820.0 N52H, N57Y, 3676939 8.4 3234 19.7 5495.2 4081.6 1442.8 Q100R, L102R, V110D, H115R,C198R N52S, F120S, I143V, 325 6567 7.9 717 4.4 2145.4 2185.7 646.1 I224VN52S, N194D 366 5690 6.8 272 1.7 2315.1 1485.0 1140.6 V11E, N30D, N52H,308 5345 6.4 1152 7.0 2747.0 3383.4 1701.2 N57Y, H94E, L96I, L98F,N194D, V210A, I218T N52S, E90A 323 5097 6.1 706 4.3 5019.8 3036.4 1482.4N52H, N57Y, 317 4737 5.7 520 3.2 2501.5 1632.1 937.5 V110A, C198R, R221IN52H, Q100R 285 4122 5.0 1466 8.9 5782.1 2861.4 967.5 N30D, K42E, N52S324 4080 4.9 273 1.7 1336.8 1260.7 541.1 N52H, N57Y, Q100P 113 3344 4.0229 1.4 2525.4 2439.5 1233.9 N52H, N57Y, 364 3064 3.7 1471 9.0 2699.52629.9 678.2 Q100R, V110D, C198R, S212G N52Y, N57Y, 112 2177 2.6 200 1.21889.5 1757.9 808.8 F138L, L203P N52H, I143T 135 1906 2.3 138 0.8 1417.11367.9 275.2 WT ICOSL 32 831 1.0 164 1.0 558.8 377.7 152.0 N52D 111 880.1 231 1.4 1288.9 1737.9 289.0

D. Mixed Lymphocyte Reaction for Assessment of Bioreactivity Suppression

Modulation of T cell activity by fusion variants was determined in amixed lymphocyte reaction (MLR) substantially as described in Example 6.Human monocytes were incubated for 6 days in the presence of IL-4 andGM-CSF and matured to dendritic cells with the additional of LPS for thefinal 24 hours. 1×10⁴ dendritic cells and 1×10⁵ human CFSE- labeled Tcells were plated per well and incubated for 4 days in the presence ofthree different concentrations (40 nM, 13.3 nM or 4.4 nM) of wildtype orrecombinant variant ICOSL ECD-Fc molecules diluted in PBS. The sameconcentrations of human IgG, PD-L2-Fc or Belatacept (CTLA4-Fc containingL104E and A29Y mutations)were used as controls. Supernatants wereharvested and IFN-gamma responses were characterized by ELISA.

FIG. 5 depicts the IFN-gamma production under the various conditions.The levels of IFN-gamma produced by cells in the presence of wild-typeICOSL is set forth by the horizontal line. No suppression of IFN-gammaproduction was observed in the presence of negative control proteinPD-L2-Fc. In contrast, most of the tested ICOSL variants exhibited somedegree of inhibition of IFN-gamma production in the MLR. Certainvariants exhibited substantial inhibition of IFN-gamma with very low tono detectable IFN-gamma produced in the cultures, even at the lowestconcentration of 4.4 nM tested. The percent MLR suppression in thepresence of 4.4 nM of variant of variant ECD ICOSL-Fc is set forth inTable 23. In the Table, the negative values indicate an inflammatoryeffect in the assay.

TABLE 23 Costimulatory bioactivity data for ICOSL in MLR SEQ ID % MLR NOSuppresion ICOSL mutation(s) (ECD) (4.4 nM) N52H, N57Y, Q100R, C198R 36593.6 N52H, N57Y, Q100R, V122A 290 94.4 N52H, N57Y, Q100R, F172S 291100.0 N52Y/N57Y/F138L/L203P 112 100.0 V11E, N30D, N52H, N57Y, H94E,L96I, L98F, 308 100.0 N194D, V210A, I218T N52H, N57Y, Q100R, L102R,V110D, H115R, 367 100.0 C198R N52H, N57Y, Q100R 283 98.2 N52H, Q100R 28597.5 N52H, N57Y, Q100R, V110D, C198R, S212G 364 90.4 N52H, N57Y, Q100P113 100.0 E16V, N52H, N57Y, Q100R, V110D, H115R, 300 100.0 Y152C, K156M,C198R N30D, K42E, N52S 324 −38.8 N52S, F120S, I143V, I224V 325 −44.2N52S, E90A 323 −30.4 N52H, N57Y, V110A, C198R, R221I 317 100.0 N52S,N194D 366 −22.3 N52H, I143T 135 −78.0 N52D 111 0.5

E. Assessment of Proliferation and Intracellular Cytokine Markers byFlow Cytometry

Carboxyfluorescein succinmidyl ester (CFSE) labeled pan T cells from MLRstudies as described above that had been incubated for 4 days in thepresence of of wildtype or recombinant variant ICOSL ECD-Fc moleculeswere further tested for cytokine levels by restimulation with phorbolmyristate acetate (PMA)/Ionomycin for 6 hours in the presence of golgiinhibitor (Golgi/Block/Plug). T cells from the MLR study that had beenincubated with human IgG, anti-CD28, anti-ICOSL, PD-L2-Fc, or Belatacept(CTLA4-Fc containing L104E and A29Y mutations) also were restimulated. Tcells were stained for CD4 or CD8 surface markers, fixed, permeabilized,and intracellularly stained for various cytokines as set forth in Table24 and 25.

The percent (%) of CD4+and CD8+T cells that were positive for specificintracellular cytokines are shown in Table 24, respectively. The resultsshowed that a number of the variant ICOSL ECD-Fc molecules were able tosuppress one or more cytokines, including, in some cases, a majority ofcytokines. A total score and mean score are calculated to assess the sumeffects of individual molecules tested over the parameters examined inthis assay. Proliferation was also assessed and a percentage of cellsthat have divided as determined by CFSE dilution is also shown in Table24 and 25. Among the provided results, the results show that certainvariants show comparable or better activity than Belatacept,particularly from the CD8+ cells.

TABLE 24 Assessment of Proliferation and Intracellular Cytokine levelsof CD4+ T cells Variant SEQ ID NO (ECD) % Cytokine+ Total Mean orProtein Prolif % IFNg+ % IL4+ % IL21+ % IL22+ % TNF+ % IL2+ % IL10+ %IL17A+ Score Score 308 3.0 9.9 3.0 1.0 1.3 34.9 31.3 0.1 0.2 41.0 4.6300 2.7 11.1 3.4 1.1 1.4 38.0 35.0 0.0 0.1 63.0 7.0 317 2.9 10.9 3.3 1.11.5 37.3 34.1 0.1 0.2 65.0 7.2 291 3.2 8.1 2.5 0.6 1.1 27.9 24.1 0.9 1.366.0 7.3 283 3.3 9.2 3.0 0.8 1.4 31.1 26.3 0.8 1.3 70.0 7.8 364 3.4 10.93.3 1.0 1.6 36.5 32.3 0.5 0.9 89.0 9.9 390 3.6 9.5 3.1 0.9 1.5 33.8 29.40.8 1.4 92.0 10.2 367 2.8 12.0 3.5 1.1 1.6 40.9 38.5 0.1 0.3 92.0 10.2CTLA-4-Ig: 10.7 10.5 2.7 2.4 2.0 24.5 19.6 0.6 1.4 99.0 11.0 L104E, A29Y(Belatacept) 112 3.5 12.0 3.8 1.3 1.6 41.3 36.7 0.2 0.4 109.0 12.1 2854.4 9.8 3.2 1.2 1.7 32.7 29.3 0.9 1.4 114.0 12.7 113 3.0 13.2 4.1 1.21.8 43.2 39.8 0.1 0.2 115.0 12.8 365 3.6 11.3 3.9 1.0 2.0 39.1 34.6 0.81.3 118.0 13.1 WT ICOSL 12.7 16.7 5.8 4.9 4.7 36.2 29.2 0.2 0.4 127.014.1 113 3.5 12.3 3.9 1.1 1.8 39.2 37.5 0.4 0.6 127.0 14.1 366 10.9 16.07.2 4.9 5.4 40.6 33.0 0.1 0.2 135.0 15.0 321 10.6 16.7 6.0 4.3 5.0 41.337.3 0.2 0.3 146.0 16.2 mlgG ctl 15.5 15.6 5.9 5.2 4.6 31.7 26.0 0.4 1.7146.0 16.2 PDL2 12.3 17.9 5.7 4.7 5.2 41.4 36.0 0.3 0.6 163.0 18.1 32311.9 17.7 6.2 5.2 5.3 42.4 37.5 0.2 0.4 167.0 18.6 WT ICOSL 12.8 17.46.3 5.4 5.6 38.9 32.1 0.3 0.6 167.0 18.6 Anti-ICOSL 15.5 16.3 6.5 5.25.5 35.1 29.1 0.7 2.0 168.0 18.7 135 12.6 17.4 6.5 5.4 4.9 44.3 37.2 0.40.5 179.0 19.9 HuIgG 12.7 17.1 6.3 5.9 4.9 41.1 32.4 0.7 1.3 181.0 20.1Anti-CD28 88.2 42.9 5.0 5.8 5.4 43.5 25.5 0.4 1.4 183.0 20.3 325 12.718.7 6.5 5.4 5.6 44.2 40.0 0.1 0.3 186.0 20.7 111 13.7 18.3 6.8 5.9 6.142.1 35.2 0.3 0.5 194.0 21.6

TABLE 25 Assessment of Proliferation andIntracellular Cytokine levels ofCD8+ T cells Variant SEQ ID NO (ECD) % Cytokine+ Total Mean or ProteinProlif % IFNg+ % IL4+ % IL21+ % IL22+ % TNF+ % IL2+ % IL10+ % IL17A+Score Score 308 4.2 8.4 1.6 2.2 47.2 7.6 8.0 0.2 0.1 67.0 7.4 300 3.88.5 2.0 2.2 47.2 8.1 9.1 0.1 0.0 69.0 7.7 317 3.9 8.8 2.0 1.7 45.6 8.49.0 0.1 0.1 64.0 7.1 291 3.8 7.0 1.4 1.8 46.6 5.9 5.9 1.1 1.1 78.0 8.7283 4.3 8.4 1.9 1.7 50.2 7.1 6.6 1.1 1.1 98.0 10.9 364 4.1 9.0 1.8 1.846.4 8.2 8.5 0.7 0.8 87.0 9.7 390 4.1 7.9 1.8 1.8 49.8 7.3 7.2 1.0 1.293.0 10.3 367 3.5 9.0 1.7 2.0 47.3 8.6 10.4 0.2 0.2 78.0 8.7 CTLA-4-Ig:12.3 14.5 2.0 3.2 38.1 11.2 8.8 0.9 1.6 121.0 13.4 L104E, A29Y(Belatacept) 112 4.2 9.6 1.9 1.8 40.4 9.4 9.9 0.3 0.3 81.0 9.0 285 5.49.5 2.0 2.7 44.2 7.8 8.4 1.2 1.3 112.0 12.4 113 3.7 9.5 1.9 1.3 44.4 9.410.5 0.1 0.1 62.0 6.9 365 4.1 9.6 2.3 1.8 46.8 9.0 9.3 0.9 1.0 122.013.6 ICOSL 17.2 22.3 4.9 6.4 46.4 22.0 15.7 0.4 0.7 181.0 20.1 113 4.29.5 2.0 2.1 45.6 8.9 10.7 0.5 0.5 110.0 12.2 366 14.5 19.4 5.6 4.8 48.419.2 13.8 0.1 0.2 142.0 15.8 321 13.4 18.9 4.3 5.0 46.3 18.4 15.4 0.30.6 138.0 15.3 mlgG ctl 20.2 25.0 4.4 4.1 35.5 24.6 15.8 0.7 1.8 174.019.3 PDL2 15.6 21.2 4.1 4.8 44.1 20.7 15.6 0.5 0.8 147.0 16.3 323 15.420.8 4.7 5.6 44.9 20.6 17.1 0.2 0.5 149.0 16.6 WT ICOSL 17.5 22.1 5.54.6 45.0 21.0 12.8 0.2 0.4 148.0 16.4 Anti-ICOSL 21.5 26.4 4.8 5.3 33.426.9 19.0 1.0 2.3 198.0 22.0 135 17.2 22.2 4.7 6.7 39.4 22.5 18.1 0.80.9 178.0 19.8 HuIgG 15.9 21.5 4.4 6.4 41.6 21.4 15.1 1.4 1.7 179.0 19.9Anti-CD28 60.6 44.3 3.5 2.1 38.6 32.5 16.0 1.2 1.6 182.0 20.2 325 16.522.0 4.9 5.4 44.0 21.9 18.5 0.2 0.6 161.0 17.9 111 17.7 22.8 5.3 6.145.4 22.9 16.7 0.4 0.6 183.0 20.3

Example 16 Assessment of Cytokine Production in B-T Cell Co-culture

B cells and CD4+ T cells were purified from the same donor and labeledwith CSFE and plated in 96 well plates in 1:1 cellular ratios at 5×10⁴cells of each per well. Variant ICOSL ECD-Fc fusion molecules orBelatacept were added at a final concentration of 40 nM per well. Cellswere either unstimulated or incubated with 100 ng/ml of Staphylococcusenterotoxin B (SEB), 1 μg/ml of Pokeweed Mitogen (PWM) or both for 7days at 37° C. in a final volume of 200 μl/well.

Cells were harvested and surface stained for the following B and T celllineage markers (IgM, IgD, CD38, CD138, CD27, CD19, CD4, CD3).Proliferation was assessed by flow cytometry and culture supernatantswere analyzed for IL-5, IL-13 or IL-21 cytokines using a LEGENDplexhuman Th cytokine detection kit (Biolegend, USA).

As shown in FIG. 6A, the number of dividing B cells was reduced in B/Tcell co-cultures when incubated in the presence of variant ICOSL ECDFc-fusion molecules compared to no protein controls. The degree ofantagonistic effect for the tested exemplary variants was similar toCTLA-4-Ig Belatacept (L104E, A29Y). Likewise, as shown in FIGS. 6B-6D,variant ICOSL ECD Fc-fusion molecules inhibited cytokine production inprimary human B cell/T cell coculture in vitro compared to no proteincontrols as well as cultures containing wild-type ICOSL control.Compared to Belatacept, exemplary tested variant ICOSL ECD Fc-fusionmolecules were more effective in blocking cytokine production in somecases.

Example 17 Assessment of Survival and Disease Activity inGraft-Versus-Host-Disease (GvHD) Model

Exemplary ICOSL variant ECD-Fc protein, were assessed for activity in agraft-versus-host-disease (GvHD) model. Female NGS mice (n=10 per group)were irradiated (100 rad) and administered 10 mg of gamma globulinsubcutaneously on Day -1. On Day 0, the mice received 10 million humanperipheral blood mononuclear cells (PBMCs) and intraperitoneal injectiondosing of either 100 μg of WT-ICOSL ECD Fc, a variant ICOSL ECD Fcmolecule N52H/I143T (ECD set forth in SEQ ID NO: 135), a variant ICOSLECD Fc molecule N52H/N57Y/Q100P (ECD set forth in SEQ ID NO: 113), 75 μgof Belatacept (CTLA-4-Ig L104E/A29Y; U.S. Patent Application PublicationNumber US20160271218) or saline as control. On Day 15, engrafted humanCD45+ cells were phenotyped by flow cytometry. After the study wasterminated on Day 35, endpoint measurements of survival, body weightloss, and disease activity were evaluated.

FIG. 7A shows the survival of GVHD mice treated with saline, WTICOSL-ECD Fc, the variantlCOSL ECD-Fc molecules, or Belatacept. Asignificant difference in the survival of mice administered variantICOSL ECD- Fc N52H/N57Y/Q100P (ECD set forth in SEQ ID NO: 113) comparedto mice administered saline or WT ICSOL ECD-Fc was observed (p<0.0001 byMantel-Cox and Gehan-Breslow-Wilcoxon test). FIG. 7B shows similardifferences between the body weight loss of mice treated with saline, WTICOSL ECD- Fc, the variant ICOSL ECD- Fc molecules, or Belatacept overthe course of the study.

A disease activity index (DAI) was determined three times a week duringthe study and evaluated the scoring of weight loss, posture, activity,appearance of the hair coat and skin of the mice. The grade of diseaseover the course of the study is shown in FIG. 7C. Treatment groups thatreceived ICOSL Fc variant N52H/N57Y/Q100P (ECD set forth SEQ ID NO: 113)or Belatacept showed significantly improved DAI scores. The percentageof human T cells in the peripheral blood on day 14 of the study was alsoassessed by flow cytometry. Measurements were averaged by treatmentgroup and error bars represent standard error of the mean (SEM). FIG. 7Dshows the percent of live CD3+/CD4+ or CD3+/CD8+ cells in the blood.Treatment groups that received variant ICOSL ECD Fc with N52H/N57Y/Q100P(ECD set forth SEQ ID NO: 113) or Belatacept showed significantlydifferent levels of CD4+ T cells compared to saline treatment group(p=0.008 and 0.006, respectively, by unpaired t-test). This studydemonstrates the therapeutic effect of the variant ICOSL Fc variantprotein on human primary T cells and GVHD during in vivo modeling.

Example 18 Assessment of Activation by Stacked Molecules

Stacked variant IgV Fc fusion proteins containing an NKp30 IgV domain asa localizing domain (designated “L”) and an ICOSL IgV domain as acostimulatory domain (designated “C”) were generated and assessedsubstantially as described in Example 8. Specifically, the constructstested in this experiment include: (1) a stacked construct with variantIgV Fc fusion protein (vIgD C-L) containing an NKp30 composed of Ig ofconsensus NKp30 variant (SEQ ID NO:493) with the IgV domain of ICOSLvariant N52H/N57Y/Q100P (ECD set forth in SEQ ID NO: 113 and IgV setforth in SEQ ID NO:201); (2) a stacked construct with the NKp30 wildtypedomain Ig domain (SEQ ID NO:214) and V domain of wildtype ICOSL (WT C-L)(SEQ ID NO:196); (3) a construct with wild-type ICOSL IgV domain (WT Cdomain; SEQ ID NO:196), and (3) a construct with the wildtype NKp30domain (WT L domain; SEQ ID NO: 214).

CD32+ K562 cells were engineered to stably express B7-H6 on the cellsurface. The cells were then treated with mitomycin and plated with panT-cells in the presence of absence of 10 nM anti-CD3 and stackedvariants or control domains at 100, 33, 11, or 3.7 nM. Cells werecultured 3 days before harvesting culture supernatants and measuringhuman IFN-gamma levels using an ELISA.

As shown in FIG. 8, both variant and wildtype costimulatory-localizingdomain stacks were able to localize to the engineered K562 cells anddeliver a co-stimulatory signal to the pan-T-cells to induce IFN-gammasecretion. The stacked construct with variant IgV Fc fusion protein(vIgD C-L) showed increased functional activity results at allconcentrations tested, while the individual domain components had noeffect when not combined with each other.

Example 19 Assessment of Delayed Type Hypersensitivity In Vivo

Variant ICOSL ECD-Fc fusion molecules were assessed foranti-inflammatory activity in vivo in the mouse delayed-typehypersensitivity (DTH) model. Delayed-type hypersensitivity immunereactions were elicited in ovalbumin (OVA)-sensitized mice and responseafter challenge was assessed. The variant ICOSL ECD-Fc fusion moleculestested contained a variant ECD with the following amino acidsubstitutions: N52H/N57Y/Q100P (ECD set forth in SEQ ID NO: 113),N52H/Q100R (ECD set forth in SEQ ID NO: 285), or N52H/N57Y/Q100R/F172S(ECD set forth in SEQ ID NO: 291). The variants were fused to either anFc backbone containing mutations C220S/L234A/L235E/G237A by EU numbering(designated Fc#1) set forth in SEQ ID NO:477) or an Fc backbonecontaining mutations C220S/E233P/L234V/L235A/G236del/S267K by EUnumbering (designated Fc#2) set forth in SEQ ID NO:478) either with orwithout a G4S (GGGGS) linker. Table 26 sets forth the tested constructs:

TABLE 26 ICOSL ECD-Fc Fusion Constructs ICOSL ECD (SEQ G4S Fc ID NO)Linker (SEQ ID NO) N52H/N57Y/Q100P (G4S)-Fc 113 + 477 #1 N52H/Q100R(G4S)-Fc #1 285 + 477 N52H/N57Y/Q100R/F172S 291 + 478 (G4S)-FcN52H/N57Y/Q100R/F172S 291 + 477 (G4S)-Fc N52H/N57Y/Q100R/F172S-Fc 291 −477

For sensitization, 8-week old female BALB/c mice were injectedsubcutaneously with 100 μg of OVA emulsified in Sigma Adjuvant (100 μL;catalog number 56322-1VL) at the base of the tail on day 0. On days 1and 4, the mice were administered variant ICOSL ECD-Fc fusion proteins,75 μg of CTLA-4 Fc (abatacept), or PBS as negative control byintraperitoneal injection. On day 7 at two to three hours prior to OVAchallenge, the mice were further administered PBS as a control, 75 μg ofCTLA-4 Fc (abatacept from Orencia), or the indicated variant ICOSLpolypeptide by intraperitoneal injection. Abatacept and variant ICOSL-Fcfusion molecules were dosed at molar equivalents.

For OVA challenge, an intradermal injection of 10 μg OVA in the left earpinnae in a 10 μL volume of PBS was delivered under gas isofluraneanesthesia 2-3 hours following therapeutic treatment. Baseline earthickness was measured prior to OVA challenge.

On day 8, ear thickness was measured under isoflurane anesthesia usingMitutoyo calipers and the change in ear thickness before and after OVAchallenge was determined. As shown in FIG. 9, mice treated with theindicated variant ICOSL ECD-Fc fusion molecules showed significantlyless OVA-induced ear swelling as compared to PBS control (<0.0001 by1-way ANOVA). There were no significant differences between earthickness for mice treated with abatacept compared to any of theindicated variant ICOSL ECD-Fc fusion molecules tested or between thevariant ICOSL treatments. These results demonstrate that variant ICOSLmolecules can reduce immune responses in vivo.

Example 20 Generation and Assessment of Binding and Activity of VariantICOSL IgSF Domain-Containing Molecules

Additional variant ICOSL IgSF (e.g. ECD) domain-containing moleculeswere generated, as described below. In each of the Tables below, theTable indicates amino acid substitutions in the ECD of the variant ICOSLas designated by amino acid position number corresponding to amino acidpositions in the respective reference unmodified ICOSL extracellulardomain (ECD) sequence set forth in SEQ ID NO:32. In some cases, theremoval of the AAA linker sequence of the variant ICOSL ECD-Fc isindicated by “AAAA”. Column 2 sets forth the SEQ ID NO identifier foreach variant ECD domain contained in the variant ECD-Fc fusion molecule.

A. Generation of Additional Variants 1. Solubility Variants

From a collection of mutants containing the following mutationsincluding E16V, N30D, K42E, N52H, N52Y, N52S, N57Y, E90A, Q100R, Q100P,L102R, V110D, H115R, F1205, V122A, F138L, I143V, I143T, H152C, K156M,F172S, N194D, C198R, L203P, R221I, I224V, the mutations H115R, F172S andC198R were identified as mutations that may potentially enhance proteinsolubility or enhance protein expression (‘solubility mutations’). Thesethree mutations (H115R, F172S and C198R) were randomly introduced bysite directed mutagenesis into the same set of clones to generate acollection of derivatives that contain one or more of these solubilitymutations. Because site directed mutagenesis reaction was carried outwith pooled mutagenic oligos reacted with pooled parental clones in asingle reaction, some of the clones also contain some mutations fromother parental clones. The generated variants contained between 3 to 10different amino acid mutations in various combinations, as summarized inTable 27A.

TABLE 27A Exemplary variant ICOSL polypeptides ECD SEQ ID Mutation(s) NOWild-type 32 N52H/N57Y/Q100R/H115R/C198R 435 N52H/N57Y/Q100R/F172S/C198R436 N52H/N57Y/Q100R/H115R/F172S/C198R 437N52H/N57Y/Q100R/H115R/I143V/F172S/C198R 438N52H/N57Y/Q100R/L102R/H115R/F172S/C198R 439 N52H/V122A/F172S/C198R 440N52H/N57Y/Q100R/H115R/F172S/N194D 441 N52H/N57Y/H115R/F172S/C198R 442N52H/N57Y/Q100R/H115R/C198R 443 N52H/N57Y/H115R 444N52H/N57Y/Q100R/H115R 445 N52H/N57Y/Q100R/H115R/F172S/I224V 446N52H/N57Y/Q100R/H115R/F172S 447 N52H/N57Y/Q100R/F172S 448N52H/Q100R/H115R/I143T/F172S 449 N52H/N57Y/Q100P/H115R/F172S 450N52Y/N57Y/Q100P/F172S 451 E16V/N52H/N57Y/Q100R/V110D/H115R/C198R 452E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/ 453 K156M/F172S/C198RN52S/E90A/H115R 454 N30D/K42E N52S/H115R 455N30D/K42E/N52S/H115R/C198R/R221I 456 N30D/K42E/N52S/H115R/C198R 457N30D/K42E/N52S/H115R/F172S/N194D 458 N52S/H115R/F120S/I143V/C198R 459N52S/H115R/F172S/C198R 460 N52H/N57Y/Q100P/C198R 461 N52H/N57Y/Q100PH115R/F172S/C198R 462 N52H/N57Y/Q100P/F172S/C198R 463N52H/N57Y/Q100P/H115R 464 N52H/N57Y/Q100P/H115R/C198R 465N52H/Q100R/C198R 466 N52H/Q100R/H115R/F172S 467N52H/Q100R/H115X/F172S/C198R 468 N52H/Q100R/H115R/F172S/C198R 469N52H/N57Y/Q100R/F172S/C198R 470

2. Back Variants

Particular exemplary mutations including N52H, N52Y, N57Y, Q100R, Q100P,F138L, C198R, L203P identified in select variants described in Example 6were further combined in the ECD backbone of wild-type ICOSL withreference to positions set forth in SEQ ID NO:32 to generate additionalcombination variants. The generated variants contained between 1 to 3different amino acid mutations in various combinations as set forth inTable 27B.

TABLE 27B Exemplary variant ICOSL polypeptides ECD SEQ Mutation(s) ID NOWild-type 32 Q100R 427 F138L/L203P 428 N52Y/F138L/L203P 429N57Y/Q100R/C198R 430 N57Y/F138L/L203P 431 N52H 110 N57Y 121 N57Y/Q100P122 Q100R/F138L 432 L203P 433

3. Glycosylation Variants

Exemplary glycosylation mutations selected from N52H, N52Q, N84Q, N119Q,N155H, N155Q, N168Q, N207Q were combined in various permutations in theECD backbone of wild-type ICOSL with reference to positions set forth inSEQ ID NO:32 to generate additional combination variants. The generatedvariants contained between 1 to 5 different amino acid mutations invarious combinations as set forth in Table 27C. Mutations designatedwith an “X” indicate either an N or Q at the indicated position.

TABLE 27C (glyc) Exemplary variant ICOSL polypeptides ECD SEQMutation(s) ID NO Wild-type 32 N84Q 387 N119Q 388 N168Q 389 N207Q 390N52Q/N207X 391 N168X/N207X 392 N52Q/N168Q 393 N84Q/N207Q 394 N155Q/N207Q395 N119Q/N168Q 396 N119Q/N207Q 397 N119Q/N155X 398 N52Q/N84Q 399N52Q/N119Q 400 N84Q/N119Q 401 N52Q/N84Q/N168Q 402 N52Q/N84Q/N207Q 403N84Q/N155Q/N168Q 404 N84Q/N168Q/N207Q 405 N84Q/N155H/N207Q 406N155Q/N168Q/N207Q 407 N119Q N155Q/N168Q 408 N119Q/N168Q/N207Q 409N84Q/N119Q/N207Q 410 N119Q/N155H/N207Q 411 N84Q/N119Q/N155Q 412N52Q/N119Q/N155Q 413 N52H/N84Q/N119Q 414 N52H/N84Q/N168X/N207X 415N52Q/N84Q/N155X/N168X 416 N52Q/N84Q/N119Q/N168Q 417N84Q/N119Q/N155Q/N168Q 418 N84Q/N155Q/N168Q/N207Q 419N84Q/N119Q/N155Q/N207Q 420 N52Q/N84Q/N119Q/N207Q 421N52Q/N84Q/N119Q/N155Q 422 N52Q/N84Q/N119Q/N155Q/N207Q 423N84Q/N119Q/N155Q/N168Q/N207Q 424

B. Binding to Cell-Expressed Counter Structures

The additional variants were formatted as Fc-fusion proteins asdescribed in Example 4. The variant Fc-fusion molecules were assessed inbinding studies to assess specificity and affinity of ICOSL domainvariant immunomodulatory proteins for cognate binding partners. Expi293cells transfected with cognate binding partners, human CD28, ICOS andCTLA4, were used in binding studies as described in Example 6. MFI wasdetermined for each transfectant and compared to the correspondingunmodified (parental) ECD-Fc.

Results for the binding for exemplary variant ICOSL ECD-Fc fusionmolecules are shown in Tables 28A-C. As shown in Table 28A-C, ICOSL IgSF(e.g. ECD) domain variants generated with the various combinations ofspecific mutations exhibited increased binding for at least one, and insome cases more than one, cognate counter structure ligand.

C. Bioactivity Characterization with Anti-CD3 Coimmobilization Assay

The costimulatory bioactivity of generated variant Fc-fusion moleculeswas also assessed in anti-CD3 coimmobilization assays as described inExample 6. Table 28A-C depicts the ratio of IFN-gamma produced by eachvariant ECD-Fc compared to the corresponding unmodified (wildtype) ICOSLECD-Fc in the assay. Mutations designated with an “X” indicate a Q orthe wildtype residue corresponding to the indicated position of SEQ IDNO: 32 at the indicated position. As shown, variant Fc-fusion moleculesgenerated exhibited improved activities to increase immunologicalactivity.

TABLE 28A Molecule sequences, binding data, and costimulatorybioactivity data of variant ICOSLECD-Fc molecules containing selectmutations Coimmobilization with anti- Binding CD3 ICOS CD28 CTLA-IFN-gamma SEQ ID MFI MFI 4 MFI pg/mL NO (parental (parental (parental(parental ICOSL Mutations (ECD) ratio) ratio) ratio) ratio) N52H, N57Y,Q100R, F172S, 436 118145 59651 178790 5059 (37.90) C198R (1.33) (29.60)(41.12) N52H, N57Y, Q100R, H115R, 437 125341 51604 211000 8218 (61.57)F172S, C198R (1.41) (25.60) (48.53) N52Y, N57Y, Q100P, F172S 451 12128063663 174224 8123 (60.86) (1.37) (31.59) (40.07) E16V, N52H, N57Y,Q100R, 453 107819 68883 170080 8936 (66.95) V110D, H115R, Y152C, (1.22)(34.18) (39.12) K156M, F172S, C198R N52S, H115R, F120S, I143V, 459116235 25582 22483 125 (0.93) C198R (1.31) (12.69) (5.17) N52H, N57Y,Q100P, C198R 461 107164 56103 172319 1258 (9.43)  (1.21) (27.84) (39.63)N52H, N57Y, Q100P, H115R, 462 120864 54586 176637 5507 (41.26) F172S,C198R (1.36) (27.08) (40.63) N52H, N57Y, Q100P, F172S, 463 117954 59376151265 3884 (29.10) C198R (1.33) (29.46) (34.79) N52H, N57Y, Q100P,H115R 464 126221 53321 178812 4154 (31.13) (1.42) (26.46) (41.13) N52H,N57Y, Q100P, H115R, 465 137004 55454 148417 5069 (37.98) C198R (1.55)(27.51) (34.14) N52H, Q100R, C198R 466 111428 58608 116111 3729 (27.94)(1.26) (29.08) (26.71) N52H, Q100R, H115R, F172S 467 105532 58287 1062955294 (39.67) (1.19) (28.92) (24.45) N52H, Q100R, H115X, 468 106555 73397171815 6961 (52.16) F172S, C198R (1.20) (36.42) (39.52) N52H, Q100R,H115R, 469 114223 66686 157154 7592 (56.88) F172S, C198R (1.29) (33.09)(36.15) N52H, N57Y, Q100R, F172S, 470 99350 61292 182288 9167 (68.68)C198R (1.12) (30.41) (41.93) N52H, N57Y, Q100R, H115R, 437 114057 52011146471 6545 (49.04) F172S, C198R (1.29) (25.81) (33.69) N52H, N57Y,Q100R, H115R, 447 136143 66516 177376 8527 (63.89) F172S (1.54) (33.00)(40.80) N52H, N57Y, Q100R, H115R, 437 132970 59633 133247 5999 (44.95)F172S, C198R (1.50) (29.59) (30.65) Q100R 427 62064 16740 29654  35(0.26) (8.31) (8.31) (8.31) Q100R ΔAAA 427 1594 16535 33457  87 (0.65)(8.20) (8.20) (8.20) F138L L203P 428 53804 1510 2151  35 (0.26) (0.75)(0.75) (0.75) F138L L203P ΔAAA 428 53044 1882 1623  35 (0.26) (0.93)(0.93) (0.93) N52Y F138L L203P 429 99761 47369 67300 1489 (11.16)(23.50) (23.50) (23.50) N52Y F138L L203P ΔAAA 429 59576 52865 66553 997(7.47) (26.23) (26.23) (26.23) N57Y Q100R C198R 430 58706 57739 994269962 (74.64) (28.65) (28.65) (28.65) N57Y Q100R C198R 430 98514 57694131458 6763 (50.67) ΔAAA (28.63) (28.63) (28.63) N57Y F138L L203P 431109472 42276 64477 4979 (37.30) (20.98) (20.98) (20.98) N57Y F138L L203PΔAAA 431 97777 44924 64742 6507 (48.75) (22.29) (22.29) (22.29) N52H 11091598 58264 103025 3393 (25.42) (28.91) (28.91) (28.91) N57Y 121 10903143754 50683 4881 (36.57) (21.71) (21.71) (21.71) N57Y, Q100P 122 7248060161 109522 2797 (20.95) (29.85) (29.85) (29.85) Q100R, F138L 432 659744485 8136 685 (5.13) (2.23) (2.23) (2.23) L203P 433 61554 1533 2031 2434(18.24) (0.76) (0.76) (0.76) Wildtype ICOSL ECD 32 88625 2015 4348 133(1.00) (1.00) (1.00) (1.00)

TABLE 28B Molecule sequences, binding data, and costimulatorybioactivity data of variant ICOSLECD-Fc molecules containing selectmutations Coimmobilization with anti- Binding CD3 ICOS CD28 CTLA-IFN-gamma SEQ MFI MFI 4 MFI pg/mL ID NO (parental (parental (parental(parental ICOSL Mutations (ECD) ratio) ratio) ratio) ratio) N52H, N57Y,Q100R, H115R 445 165027 51666 287581 5858 (20.36) (1.97) (9.89) (60.27)N52H, N57Y, Q100R, F172S 448 184449 51394 182109 3449 (11.99) (2.20)(9.84) (38.16) N52H, N57Y, Q100R, H115R, 446 165120 46636 274026 2053(7.13)  F172S, I224V (1.97) (8.93) (57.43) N52H, N57Y, Q100R, H115R, 447164750 40046 259351 3722 (12.93) F172S (1.97) (7.67) (54.35) N52H, N57Y,Q100R, H115R, 435 186017 39073 200505 3909 (13.58) C198R (2.22) (7.48)(42.02) N52H, N57Y, Q100R, F172S, 436 181118 38233 210709 1199 (4.17) C198R (2.16) (7.32) (44.16) N52H, N57Y, Q100R, H115R, 437 155392 28828169736 3449 (11.99) F172S, C198R (1.85) (5.52) (35.57) N52H, N57Y,Q100R, H115R, 438 139977 31459 179089 1620 (5.63)  I143V, F172S, C198R(1.67) (6.02) (37.53) N52H, N57Y, Q100R, L102R 439 146799 29636 2000002712 (9.43)  H115R, F172S, C198R (1.75) (5.68) (41.91) N52H, N57Y,Q100R, H115R 441 150863 31304 167783 15607 (54.24)  F172S, N194D (1.80)(5.99) (35.16) N52H, N57Y, H115R, F172S, 442 126909 35803 152858 5374(18.67) C198R (1.51) (6.86) (32.03) N52H, N57Y, Q100R, H115R, 443 13173037595 139041 9306 (32.34) C198R (1.57) (7.20) (29.14) N52H, N57Y, H115R444 162632 49847 266878 2918 (10.14) (1.94) (9.55) (55.93) N52H, Q100R,H115R, I143T 449 132873 52058 186366 3086 (10.72) F172S (1.59) (9.97)(39.06) N52H, N57Y, Q100P, H115R, 450 148160 46851 246636 4987 (17.33)F172S (1.77) (8.97) (51.69) E16V, N52H, N57Y, Q100R, 452 154036 48674212905 5095 (17.71) V110D, H115R, C198R (1.84) (9.32) (44.62) N52S,E90A, H115R 454 142963 3597 3772 2241 (7.79)  (1.71) (0.69) (0.79) N30D,K42E, N52S, H115R, 456 124095 8066 7751 417 (1.45) C198R R221I (1.48)(1.54) (1.62) N30D, K42E, N52S, H115R, 457 161734 2791 2919 841 (2.92)C198R (1.93) (0.53) (0.61) N30D, K42E, N52S, H115R, 458 117880 4395 49412904 (10.09) F172S, N194D (1.41) (0.84) (1.04) N30D, K42E, N52S, H115R,455 114107 2935 2748 549 (1.91) (1.36) (0.56) (0.58) N52S, E90A, H115R,454 120450 12768 23282 2890 (10.04) (1.44) (2.45) (4.88) N30D, K42E,N52S, H115R 455 115273 11964 22779 2241 (7.79)  (1.38) (2.29) (4.77)N52S, H115R, F172S, C198R 460 95537 7614 21701 1458 (5.07)  (1.14)(1.46) (4.55) Wildtype 32 83813 5222 4772 288 (1.00) (1.00) (1.00)(1.00)

TABLE 28C Molecule sequences, binding data, and costimulatorybioactivity data of variant ICOSLECD-Fc molecules containingglycosylation mutations Coimmobilization with anti- Binding CD3 ICOSCD28 CTLA- IFN-gamma SSEQ MFI MFI 4 MFI pg/mL ID NO (parental (parental(parental (parental ICOSL Mutation(s) (ECD) ratio) ratio) ratio) ratio)N84Q 387 34426 1755 5757 100 (2.03)  (0.94) (1.16) (1.51) N119Q 38830806 4102 19836 81 (1.66) (0.84) (2.70) (5.21) N168Q 389 27041 141018641 67 (1.36) (0.74) (0.93) (4.90) N207Q 390 36516 11923 25701 206(4.20)  (1.00) (7.86) (6.76) N52Q, N207X 391 30216 12086 27952 77 (1.56)(0.83) (7.97) (7.35) N168X, N207X 392 37191 5787 12280 104 (2.12) (1.02) (3.81) (3.23) N52Q, N168Q 393 32576 12638 27167 101 (2.06) (0.89) (8.33) (7.14) N84Q, N207Q 394 37176 5292 3153 31 (0.63) (1.02)(3.49) (0.83) N155Q, N207Q 395 34884 1489 987 73 (1.48) (0.95) (0.98)(0.26) N119Q, N168Q 396 29099 2534 11289 51 (1.05) (0.80) (1.67) (2.97)N119Q, N207Q 397 32603 1861 6795 153 (3.12)  (0.89) (1.23) (1.79) N119QN155X 398 38516 15318 27498 173 (3.52)  (1.05) (10.10) (7.23) N52Q, N84Q399 33988 1675 3525 39 (0.80) (0.93) (1.10) (0.93) N52Q, N119Q 400 3572911040 26139 51 (1.03) (0.98) (7.28) (6.87) N84Q, N119Q 401 34777 14932877 39 (0.80) (0.95) (0.98) (0.76) N52Q, N84Q, N168Q 402 27021 1584 95838 (0.78) (0.74) (1.04) (0.25) N52Q, N84Q, N207Q 403 39942 13396 2636037 (0.76) (1.09) (8.83) (6.93) N84Q, N155Q, N168Q 404 27812 357 466 30(0.61) (0.76) (0.24) (0.12) N84Q, N168Q, N207Q 405 30659 737 861 25(0.52) (0.84) (0.49) (0.23) N84Q, N155H, N207Q 406 13557 685 607 29(0.59) (0.37) (0.45) (0.16) N155Q, N168Q, N207Q 407 13999 277 317 40(0.82) (0.38) (0.18) (0.08) N119Q, N155Q, N168Q 408 36896 4094 2179 50(1.02) (1.01) (2.70) (0.57) N119Q, N168Q, N207Q 409 29543 921 3744 72(1.47) (0.81) (0.61) (0.98) N84Q, N119Q, N207Q 410 21357 569 640 59(1.20) (0.58) (0.38) (0.17) N119Q, N155H, N207Q 411 37310 614 931 86(1.75) (1.02) (0.40) (0.24) N84Q, N119Q, N155Q 412 2675 262 291 34(0.70) (0.07) (0.17) (0.08) N52Q, N119Q, N155Q 413 27853 552 772 42(0.87) (0.76) (0.36) (0.20) N52H, N84Q, N119Q 414 40700 4580 4601 39(0.80) (1.11) (3.02) (1.21) N52H, N84Q, N168X, N207X 415 8796 587 481 32(0.66) (0.24) (0.39) (0.13) N52Q, N84Q, N155X, N168X 416 43521 6605 481132 (0.66) (1.19) (4.35) (1.26) N52Q, N84Q, N119Q, N168Q 417 39342 45193300 37 (0.76) (1.07) (2.98) (0.87) N52Q, N84Q, N119Q, N207Q 421 7011602 433 37 (0.75) (0.19) (0.40) (0.11) Wildtype ICOSL ECD 32 36602 15173804 49 (1.00) (1.00) (1.00) (1.00)

Example 21 Generation and Assessment of Fusion Molecules withHER2-Targetting Antibody

This Example describes the generation and assessment of variant ICOSLECD-Fc fusion molecules conjugated with a tumor targeting agent to forma conjugate (“vIgD conjugate”).

The V-domain only of the ICOSL vIgD (N52H/N57Y/Q100P; set forth in SEQID NO:201) was fused to the amino and carboxyl termini of the lightchain (FIG. 10A) and heavy chain (FIG. 10B) of a HER2 targeting antibodywith intervening GGGSGGGS linkers. Exemplary configurations of vIgDconjugates are shown in FIG. 10C.

To assess HER2 binding, HER2 DNA or mock Expi293 transfectants werestained with titrated amounts of a HER2 targeting antibody containingthe variant ICOSL conjugate (vIgD N52H/N57Y/Q100P conjugate) at aconcentration of 100 pM to 100 nM. Control proteins, including wildtypeICOSL ECD-Fc fusion, wildtype PD-L2 IgV-Fc fusion, and variant ICOSLECD-Fc fusion molecule with mutations at N52H/N57Y/Q100P, were alsotested. Mean Fluorescence Intensity (MFI) or percent positive cells wasdetermined for each transfectant as described in Example 6. All IgSFconjugates generated as shown in FIG. 11A-B retained binding to HER2compared to the endogenous level of HER2 expression observed in Expi293cells. Similarly, vIgD conjugates also showed binding to cognate bindingpartners of ICOSL including CD28, CTLA-4, and ICOS.

Protein bioactivity and proliferation of human primary T cell in vitroassays were also characterized as described in Example 6. vIgDconjugates were bound overnight to 96-well plates at 30-0.1 nM in thepresence of 10 nM anti-CD3. The plates were washed and 100,000CFSE-labeled pan T cells were added to the plates and incubated for 72hours. IFN gamma levels in supernatant were assayed by ELISA. As shownin FIG. 12, vIgD conjugates with the indicated configurations showedgreater IFN gamma secretion and proliferation compared to parentalwild-type ICOSL ECD-Fc fusion molecule conjugate.

Example 22 Nanostring Transcriptional Signature of Primary Human T Cells

Tissue culture plates were coated with 10 nM anti-CD3 with 40 nM of anFc-control protein, wild-type ICOSL-Fc, wild-type CD80-Fc, both of theseproteins, or a variant ICOSL Fc-fusion proteins with mutations asindicated. Purified human T cells were then plated on the protein coatedplates and incubated at 37° C. Cultures from each treatment groupdescribed above were harvested at 24, 48 and 72 hours and total RNA wasprepared from each cell sample. The RNA was transferred to Nanostringand a Cancer Immune chip was used to quantitate transcripts of 750 genein each sample. Transcript values were normalized using Nanostring'sproprietary software allowing comparison of transcript levels betweentreatment groups and over the various time points. As shown in FIG. 18and FIG. 19, the variant ICOSL ECD-Fc polypeptides tested show alteredinflammatory activity compared to wildtype CD80 ECD-Fc, wildtype ICOSLECD-Fc, or a combination of both.

Example 23 Generation and Assessment of Fusion Molecules withHER2-Targetting Antibody

Proliferation of human T-cells co-cultured with VmAbs and HER2expressing target cells was also characterized. CFSE-labeled pan T-cellswere stimulated for 72 hours with K562-derived artificial target cellsdisplaying cell surface anti-CD3 single chain Fv (OKT3) and HER2 in thepresence of VmAbs or control proteins. Proliferation was measured byflow cytometric analysis of CFSE-dilution on CD4⁺ or CD8⁺ stainedT-cells. Vmabs were assayed varying either target cell number or theconcentration of the VmAb utilized. In the first assay, K562 targetcells were titrated from 2500 to 78 cells/well and added to 100,000T-cells for an effector:target (E:T) range of 40 to 1280:1. VmAbs,parental IgSF domain, or WT ICOSL were added at 1000 pM. In the secondassay, K562 target cells were added at 625 cells/well to 100,000 T-cellsfor an effector:target ratio of 160:1. VmAbs or control proteins weretitrated and added at 3000 to 37 pM. As shown in FIG. 20A and 20B, bothconfigurations of the assay demonstrate VmAbs containing thevIgD-conjugate provide superior proliferation compared to the parentalantibody, parental IgSF domain, or WT ICOSL. Additionally,vIgD-conjugates mediated proliferation at low E:T ratios (1280:1) or atlow protein concentrations (37 pM).

The present invention is not intended to be limited in scope to theparticular disclosed embodiments, which are provided, for example, toillustrate various aspects of the invention. Various modifications tothe compositions and methods described will become apparent from thedescription and teachings herein. Such variations may be practicedwithout departing from the true scope and spirit of the disclosure andare intended to fall within the scope of the present disclosure.

1-165. (canceled)
 166. A variant ICOS Ligand (ICOSL) polypeptide,comprising an IgV domain or specific binding fragment thereof, an IgCdomain or a specific binding fragment thereof, or both, wherein thevariant ICOSL polypeptide comprises one or more amino acid modificationsat one or more positions in an unmodified ICOSL or a specific bindingfragment thereof corresponding to position(s) selected from 10, 11, 13,16, 18, 20, 25, 27, 30, 33, 37, 38, 42, 43, 47, 52, 54, 57, 61, 62, 67,71, 72, 74, 75, 77, 78, 80, 84, 89, 90, 92, 93, 94, 96, 97, 98, 99, 100,102, 103, 107, 109, 110, 111, 113, 115, 116, 117, 119, 120, 121, 122,126, 129, 130, 132, 133, 135, 138, 139, 140, 142, 143, 144, 146, 148,151, 152, 153, 154, 155, 156, 158, 161, 164, 166, 168, 172, 173, 175,190, 192, 193, 194, 198, 201, 203, 207, 208, 210, 212, 217, 218, 220,221, 224, 225, or 227 with reference to SEQ ID NO:32.
 167. The variantICOSL polypeptide of claim 166, wherein the unmodified ICOSL comprises(i) the sequence of amino acids set forth in SEQ ID NO:32, (ii) asequence of amino acids that has at least 95% sequence identity to SEQID NO:32; or (iii) a portion of the sequence of (i) or (ii) comprisingan IgV domain or IgC domain or specific binding fragments thereof orboth.
 168. The variant ICOSL polypeptide of claim 166, wherein thevariant ICOSL comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20 amino acid modifications compared to theunmodified ICOSL polypeptide.
 169. The variant ICOSL polypeptide ofclaim 166, wherein the one or more amino acid modifications are selectedfrom M10V, M10I, V11E, S13G, E16V, S18R, A20V, S25G, F27S, F27C, N30D,Y33del, Q37R, K42E, T43A, Y47H, N52H, N52D, N52Q, N52S, N52Y, N52K,S54A, S54P, N57D, N57Y, R61S, R61C, Y62F, L67P, A71T, G72R, L74Q, R75Q,D77G, F78L, L80P, N84Q, E90A, K92R, F93L, H94E, H94D, L96F, L961, V97A,L98F, S99G, Q100R, Q100K, Q100P, L102R, G103E, V107A, V1071, S109G,S109N, V110D, V110N, V110A, E111del, T113E, H115R, H115Q, V116A, A117T,N119Q, F1201, S121G, V122A, V122M, F120S, S126T, S126R, H129P, S130G,S132F, Q133H, E135K, F138L, T139S, C140del, C140D, S142F, I143V, I143T,N144D, Y146C, V151A, Y152C, Y152H, W153R, I154F, N155H, N155Q, K156M,D158G, L161P, L161M, L166Q, N168Q, F172S, L173S, M175T, T190A, T190S,S192G, V193M, N194D, C198R, N201S, L203P, L203F, N207Q, L208P, V210A,S212G, D217V, I218T, 1218N, E220G, R221G, R221I, I224V, T225A, N227K, ora conservative amino acid substitution thereof.
 170. The variant ICOSLpolypeptide of claim 166, wherein the one or more amino acidmodifications are selected from among N52Y/N57Y/F138L/L203P,N52H/N57Y/Q100P, N52S/Y146C/Y152C, N52H/C198R, N52H/C140D/T225A,N52H/C198R/T225A, N52H/K92R, N52H/S99G, N57Y/Q100P, N52S/S130G/Y152C,N52S/Y152C, N52S/C198R, N52Y/N57Y/Y152C, N52Y/N57Y/ H129P/C198R,N52H/L161P/C198R, N52S/T113E, N52D/S54P, N52K/L208P, N52S/Y152H,N52D/V151A, N52H/I143T, N52S/L80P, F120S/Y152H/N201S, N52S/R75Q/L203P,N52S/D158G, N52D/Q133H, N52S/N57Y/H94D/L96F/L98F/Q100R,N52S/N57Y/H94D/L96F/L98F/Q100R/G103E/F120S, N52H/F78L/Q100R,N52H/N57Y/Q100R/V110D, N52H/N57Y/R75Q/Q100R/V110D, N52H/N57Y/Q100R,N52H/N57Y/L74Q/Q100R/V110D, N52H/Q100R, N52H/S121G,A20V/N52H/N57Y/Q100R/S109G, N52H/N57Y/R61S/Q100R/V110D/L173S,N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S, N52H/N57Y, N52S/F120S,N52S/V97A, N52S/G72R, N52S/A71T/A117T, N52S/E220G,Y47H/N52S/V107A/F120S, N52H/N57Y/Q100R/V110D/S132F/M175T,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/C198R,Q37R/N52H/N57Y/Q100R/V110N/S142F/C198R/D217V/R221G,N52H/N57Y/Q100R/V110D/C198R,N52H/N57Y/Q100R/V110D/V116A/L161M/F172S/S192G/C198R,F27S/N52H/N57Y/V110N, N52S/H94E/L961/S109N/L166Q,S18R/N52S/F93L/1143V/R221G, A20T/N52D/Y146C/Q164L,V11E/N30D/N52H/N57Y/H94E/L961/L98F/N194D/V210A/1218T,N52S/H94E/L96I/V122M, N52H/N57Y/H94E/L961/F1201/S126T/W153R/1218N,M10V/S18R/N30D/N52S/S126R/T139S/L203F, S25G/N30D/N52S/F120S/N227K,N30D/N52S/L67P/Q100K/D217G/R221K/T225S,N52H/N57Y/Q100R/V110D/A117T/T190S/C198R,N52H/N57Y/Q100R/V110D/F172S/C198R,S25G/F27C/N52H/N57Y/Q100R/V110D/E135K/L173S/C198R,N52H/N57Y/V110A/C198R/R221I,M10I/S13G/N52H/N57Y/D77G/V110A/H129P/I143V/F172S/V193M/C198R,N52H/N57Y/R61C/Y62F/Q100R/V110N/F120S/C198R,N52H/N57Y/Q100R/V110D/H115R/C198R,N52H/N57Y/Q100R/V110D/N144D/F172S/C198R, N52S/H94E/L98F/Q100R,N52S/E90A, N30D/K42E/N52S, N52S/F120S/I143V/I224V,N52H/N57Y/Q100R/V110D/C198R/S212G, N52H/N57Y/Q100R/C198R, N52S/N194D,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52S/N194D, N52S/S54P,T38P/N52S/N57D, E111del, Y33del, N52H/C140del/T225A,N52H/F78L/Q100R/C198R, N52H/N57Y/R75Q/Q100P/V110D,N52H/N57Y/L74Q/V110D/S192G, N52H/S121G/C198R, N52S/F120S/N227K,N52S/A71T/A117T/T190A/C198R, T43A/N52H/N57Y/L74Q/D89G/V110D/F172S,N52H/N57Y/Q100R/V110D/S132F/M175T, N52D,N52H/N57Y/Q100R/V107I/V110D/I154F/C198R/R221G, N52Q/N207Q, N168Q/N207Q,N52Q/N168Q, N84Q/N207Q, N155Q/N207Q, N119Q/N168Q, N119Q/N207Q,N119Q/N155Q, N52Q/N84Q, N52Q/N119Q, N84Q/N119Q, N52Q/N84Q/N168Q,N52Q/N84Q/N207Q, N84Q/N155Q/N168Q, N84Q/N168Q/N207Q, N84Q/N155H/N207Q,N155Q/N168Q/N207Q, N119Q N155Q/N168Q, N119Q/N168Q/N207Q,N84Q/N119Q/N207Q, N119Q/N155H/N207Q, N84Q/N119Q/N155Q, N52Q/N119Q/N155Q,N52H/N84Q/N119Q, N52H/N84Q, N52H/N84Q/N168Q/N207Q,N52Q/N84Q/N155Q/N168Q, N52Q/N84Q/N119Q/N168Q, N84Q/N119Q/N155Q/N168Q,N84Q/N155Q/N168Q/N207Q, N84Q/N119Q/N155Q/N207Q, N52Q/N84Q/N119Q/N207Q,N52Q/N84Q/N119Q/N155Q, N52Q/N84Q/N119Q/N155Q/N207Q,N84Q/N119Q/N155Q/N168Q/N207Q, F138L/L203P, N52Y/F138L/L203P,N57Y/Q100R/C198R, N57Y/F138L/L203P, Q100R/F138L,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/Q100R/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/I143V/F172S/C198R,N52H/N57Y/Q100R/L102R/H115R/F172S/C198R, N52H/V122A/F172S/C198R,N52H/N57Y/Q100R/H115R/F172S/N194D, N52H/N57Y/H115R/F172S/C198R,N52H/N57Y/Q100R/H115R/C198R, N52H/N57Y/H115R, N52H/N57Y/Q100R/H115R,N52H/N57Y/Q100R/H115R/F172S/I224V, N52H/N57Y/Q100R/H115R/F172S,N52H/N57Y/Q100R/F172S, N52H/Q100R/H115R/I143T/F172S,N52H/N57Y/Q100P/H115R/F172S, N52Y/N57Y/Q100P/F172S,E16V/N52H/N57Y/Q100R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/Y152C/K156M/F172S/C198R,N52S/E90A/H115R, N30D/K42E N52S/H115R, N30D/K42E/N52S/H115R/C198R/R221I,N30D/K42E/N52S/H115R/C198R, N30D/K42E/N52S/H115R/F172S/N194D,N52S/H115R/F120S/I143V/C198R, N52S/H115R/F172S/C198R,N52H/N57Y/Q100P/C198R, N52H/N57Y/Q100P/H115R/F172S/C198R,N52H/N57Y/Q100P/F172S/C198R, N52H/N57Y/Q100P/H115R,N52H/N57Y/Q100P/H115R/C198R, N52H/Q100R/C198R, N52H/Q100R/H115R/F172S,N52H/Q100R/F172S/C198R, N52H/Q100R/H115R/F172S/C198R, orN52H/N57Y/Q100R/F172S/C198R.
 171. The variant ICOSL polypeptide of claim166, wherein the one or more amino acid modifications are at one or morepositions corresponding to a position(s) selected from 52, 57, 100, 110,or
 198. 172. The variant ICOSL polypeptide of claim 171, wherein the oneor more amino acid modifications are selected from N52H, N52D, N52S,N52K, N52Y, N57Y, Q100P, Q100R, V110A, V110D, C198R, or a conservativeamino acid substitution thereof.
 173. The variant ICOSL polypeptide ofclaim 171, wherein the one or more amino acid modifications areN52H/N57Y/Q100R/C198R, N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/F172S,N52Y/N57Y/F138L/L203P,V11E/N30D/N52H/N57Y/H94E/L96I/L98F/N194D/V210A/I218T,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R, N52H/N57Y/Q100R, N52H/Q100R,N52H/N57Y/Q100R/V110D/C198R/S212G,N52H/N57Y/Q100R/L102R/V110D/H115R/C198R,E16V/N52H/N57Y/Q100R/V110D/H115R/V152C/K156M/C198R, N30D/K42E/N52S ,N52S/F120S/I143V/I224V, N52S/E90A, N52H/N57Y/V110A/C198R/R221I,N52H/N57Y/Q100P, N52H/N57Y/Q100R/V122A, N52H/N57Y/Q100R/C198R,N52S/N194D.
 174. The variant ICOSL polypeptide of claim 166, comprising:the sequence of amino acids set forth in any of SEQ ID NOS: 109-142,239, 280-325, 364-381, 387-424, 427-433, 435-470 or a specific bindingfragment thereof, or a sequence of amino acids that exhibits at least95% sequence identity to any of SEQ ID NOS: 109-142, 239, 280-325,364-381, 387-424, 427-433, 435-470 or a specific binding fragmentthereof and that contains the one or more of the amino acidmodifications; or the sequence of amino acids set forth in any of SEQ IDNOS: 197-199, 201-208, 210, 212, 240, 326-340, 382-386, 425-426, and 434or a specific binding fragment thereof, a sequence of amino acids thatexhibits at least 95% sequence identity to any of SEQ ID NOS: 197-199,201-208, 210, 212, 240,326-340, 382-386, 425-426, and 434 or a specificbinding fragment thereof and that contains the one or more of the aminoacid modifications.
 175. The variant ICOSL polypeptide of claim 166,wherein the variant ICOSL polypeptide specifically binds to theectodomain of human ICOS or human CD28 with increased affinity comparedto the binding of the unmodified ICOSL polypeptide to the ectodomain ofhuman ICOS or human CD28.
 176. The variant ICOSL polypeptide of claim175, wherein the variant ICOSL polypeptide specifically binds to theectodomain of human ICOS and the ectodomain of human CD28 each withincreased affinity compared to the binding of the unmodified ICOSLpolypeptide to the ectodomain of human ICOS and human CD28.
 177. Thevariant ICOSL polypeptide of claim 175, wherein the increased affinityto the ectodomain of human CD28 is increased more than 5-fold, 10-foldor 50-fold.
 178. The variant ICOSL polypeptide of claim 175, wherein theincreased affinity to the ectodomain of human ICOS is increased morethan 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold.
 179. The variant ICOSLpolypeptide of claim 166, wherein: the extracellular domain of ICOSL, ora portion thereof containing an IgV domain or specific-binding fragmentthereof, is the only ICOSL portion of the variant ICOSL polypeptide;and/or the variant ICOSL polypeptide lacks a transmembrane domain. 180.The variant ICOSL polypeptide of claim 179, wherein the IgV domain orspecific binding fragment thereof is the only ICOSL portion of thevariant ICOSL polypeptide.
 181. The variant ICOSL polypeptide of claim179, wherein the IgV domain is linked, directly or indirectly via alinker, to an Fc domain.
 182. The variant ICOSL polypeptide of claim 166that is a transmembrane immunomodulatory protein, wherein the variantICOSL polypeptide further comprises a transmembrane domain and/or acytoplasmic signaling domain.
 183. An immunomodulatory protein,comprising the variant ICOSL polypeptide of claim 166 linked to a secondpolypeptide comprising an immunoglobulin superfamily (IgSF) domain. 184.The immunomodulatory protein of claim 183, wherein the IgSF domain isaffinity modified and exhibits altered binding to one or more of itscognate binding partner(s) compared to the unmodified or wild-type IgSFdomain.
 185. A conjugate, comprising a variant ICOSL polypeptide ofclaim 166 linked to a targeting moiety that specifically binds to amolecule on the surface of a cell.
 186. The conjugate of claim 185,wherein the moiety is an antibody or antigen-binding fragment.
 187. Theconjugate of claim 185, wherein the cell is an immune cell or is a tumorcell.
 188. The conjugate of claim 185 that is a fusion protein.
 189. Anucleic acid molecule encoding a sequence of amino acids comprising thevariant ICOSL polypeptide of claim
 166. 190. An engineered cell,comprising a variant ICOSL polypeptide of claim 166 or a nucleic acidmolecule encoding a sequence of amino acids comprising the variant ICOSLpolypeptide of claim
 166. 191. The engineered cell of claim 190,wherein: the variant ICOSL polypeptide does not comprise a transmembranedomain and/or is not expressed on the surface of the cell; and/or thevariant ICOSL polypeptide is capable of being secreted from theengineered cell when expressed.
 192. The engineered cell of claim 190,wherein the variant ICOSL polypeptide comprises a transmembrane domainand/or is expressed on the surface of the cell.
 193. The engineered cellof claim 190, wherein the cell is an immune cell.
 194. The engineeredcell of claim 190, wherein the cell further comprises a chimeric antigenreceptor (CAR) or an engineered T cell receptor (TCR).
 195. Aninfectious agent, comprising a variant ICOSL polypeptide of claim 166 ora nucleic acid molecule encoding a sequence of amino acids comprisingthe variant ICOSL polypeptide of claim
 166. 196. The infectious agent ofclaim 195, wherein the infectious agent is a bacteria or a virus.
 197. Apharmaceutical composition, comprising the variant ICOSL polypeptide ofclaim 166; an immunomodulatory protein or conjugate comprising thevariant ICOSL polypeptide of claim 166; or an engineered cell orinfectious agent comprising the variant ICOSL polypeptide of claim 166or a nucleic acid molecule encoding the variant ICOSL polypeptide ofclaim 166, and optionally a pharmaceutically acceptable carrier.
 198. Anarticle of manufacture or kit comprising the pharmaceutical compositionof claim
 197. 199. A method of modulating an immune response in asubject, comprising administering the pharmaceutical composition ofclaim 197 to the subject.
 200. A method of modulating an immune responsein a subject, comprising administering the engineered cell of claim 190to the subject.
 201. The method of claim 199, wherein modulating theimmune response treats a disease or condition in the subject.
 202. Themethod of claim 200, wherein modulating the immune response treats adisease or condition in the subject.